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Studies of Sir2 and caloric restriction mimetic pathways in aging

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Studies of Sir2 and caloric restriction mimetic pathways in aging

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STUDIES OF SIR2 AND CALORIC RESTRICTION
MIMETIC PATHWAYS IN AGING
by
Jia Hu

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)

May 2013

Copyright 2013 Jia Hu
ii

Acknowledgments
My great gratitude goes to my advisor, Dr. Valter D. Longo, who has supported and
supervised me throughout my academic study. He provides me with unique insights and
ideas in my research and inspires me to overcome the difficulties encountered in my life
and study. His diligence sets an outstanding example for my future career. My deep
thanks also go to Dr. Min Wei, a colleague and a friend of mine, who has constantly
given me invaluable suggestions and support over the years. Without his kind help, it will
be hard for me to go through many technical problems. I am obliged to Dr.
FedericaMadia, Paola Fabrizio, Ying Li, Edoardo Parrella and Sangeeta Bardhan Cook in
Dr. Longo's laboratory. They offered me numerous advice and help in my yeast and mice
study. I also appreciated the warmth and kind-hearted assistance given by Chaiwei Cheng,
Dr. Priya Balasubramanian, Sebastian Brandhorst, Stefano di biase, Inyoung Choi as well
as former lab members Changhan Lee, Fernando Safdieand Abdoulaye Galbani.
Undergraduates Camille Amparo, Tamy Tran, Nahel Kapadia and Loomee Doo aided me
in many aspects of my projects. Huanying Ge from Dr. Lei Li’s laboratory helped me
with microarray data analysis. Yuan Zhong andYan Gan from Dr. Oscar Aparicio’s
laboratory kindly provided me with great ideas and technical supports for my yeast study.
The warm help from Dr. Finch’s laboratory is muchappreciated as well. Finally, I am
indebted to my husband, my little daughter and my parents whose love supports me to
accomplish my Ph.D dissertation.

iii

The experiments in chapter 2 were done in collaboration with the laboratories of Dr.
Rafael de Cabo, Susan Krzysik-Walker and Michael W. McBurney.

iv

Table of Contents

Acknowledgments............................................................................................................. ii

List of Tables...................................................................................................................... v

List of Figures.................................................................................................................... vi

Abstract.............................................................................................................................. ix

Chapter 1. Background and Significance............................................................................ 1
1.1. Yeast model and aging................................................................................................. 1
1.2. Sir2 and stress response in aging................................................................................. 3
1.3. Sir2 and genomic instability in aging.......................................................................... 6
1.4. Sir2 and caloric restriction in aging............................................................................. 8
1.5. Metabolic regulation in aging.................................................................................... 10

Chapter 2. Role of Sir2 in Chronological Aging ............................................................. 13
2.1. Introduction ............................................................................................................... 13
2.2. Materials and Methods............................................................................................... 18
2.3. Results........................................................................................................................ 26
2.3.1. Lack of Sir2 promotes stress resistance through Gis1 and Msn2/4..................... 26
2.3.2. Sir2 deficiency promotes genomic stability through homologous recombination
related repair..................................................................................................................... 42
2.3.3. Sir2 and Ras signaling......................................................................................... 56
2.4. Discussion.................................................................................................................. 65

Chapter 3. Role of Sirt1 in Caloric Restriction…………………..................................... 74
3.1. Introduction ............................................................................................................... 74
3.2. Materials and Methods............................................................................................... 75
3.3. Results........................................................................................................................ 77
3.4. Discussion.................................................................................................................. 83

Chapter 4. Acetic Acid is a Leucine-derived Ketone Body-like Metabolite Catabolized by
tor/s6k Mutants to Promote Chronological Longevity in S. cerevisiae…...……………. 87
4.1. Introduction ............................................................................................................... 87
4.2. Materials and Methods............................................................................................... 89
4.3. Results........................................................................................................................ 93
4.4. Discussion................................................................................................................ 118

Bibliography................................................................................................................... 124

v

List of Tables

Table 2.1. Up and Down-regulation of TIGO categories relating to transcription........... 58

Table 3.1. SIRT1 mice summary…………………………………….............................. 78

Table 3.2. Pairwise comparison of each mice group.……………….............................. 79

Table 3.3. Pathologies and abnormalities in old mice (> 24 months)..………............... 82

vi

List of Figures
Figure 2.1. Sir2 blocks extreme life span extension and reduces stress resistance in a
medium independent manner............................................................................................ 27

Figure 2.2. Thermal and Oxidative Stress Resistance and chronological life span (CLS) of
cells deficient in Sir2 and stress response transcription regulators Msn2, Msn4 and/or
Gis1 and protein kinase Rim15………...……………………………………….............. 29

Figure 2.3. Sir2 reduces the expression of genes with PDS element in their promoter
region……………………………………………………………………………............ 31

Figure 2.4. Thermal and oxidative stress resistance of cells deficient in Sch9, Sir2, Gis1
and protein kinase Rim15………………………………………………………............. 33

Figure 2.5. Gis1 over expression doesn’t increase life span.……………………............ 36

Figure 2.6. Sir2 reduces the fragmentation of Gis1 protein.............................................. 37

Figure 2.7. Sir2 does not inhibit Msn2 nucleus localization……………………............. 40

Figure 2.8. Lack of Sir2 activity promoted genomic stability during yeast chronological
aging…………………………………………………………………………….............. 42

Figure 2.9. Deletion of Sir2 promotes genomic stability in the presence of oxidative stress
and DNA alkylation treatment………………………………………………….............. 45

Figure 2.10. Msn2, Msn4 and Gis1 do not mediate the enhanced genomic protection of
Sir2 deficient strain………………………………………………………………........... 48

Figure 2.11. Homologous recombination machinery is required for the high homologous
recombination rate in cells lack of Sir2…………………………………………............ 50

Figure 2.12. Thermal and Oxidative Stress Resistance and Methyl methanesulfonate
(MMS) treatment of cells deficient in Sir2, Rad51 and Sch9……………………........... 52

Figure 2.13. Deletion of Sir2 restored chronological lifespan of rev1 Δ mutants but did
not increase mutation frequency………...……………………………………................ 55

Figure 2.14. Deletion of Sir2 further increased the life span of long-lived ras2 Δ
mutants.............................................................................................................................. 59

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Figure 2.15. The protein level of Ras2 increased with chronological aging and was
reduced by deletion of Sir2 in early stationary phase……………………………........... 61

Figure 2.16. Transcription level of Ras2……………………………………….............. 62

Figure 2.17. Immunoprecipitation does not reveal posttranslational deacetylation of
Ras2................................................................................................................................... 64

Figure 2.18. Quantification of Ras protein level under caloric restriction....................... 65

Figure 3.1. Kaplan-Meier survival curves of SIRT1
+/+
,
+/-
and
-/-
mice in ad libitum (AL)
and CR condition……………………………………………………………….............. 79

Figure 3.2. Behavioral tests of SIRT1 mice…………………………………….............. 80

Figure 3.3. CR increased SIRT1 expression is increased in response to CR……............ 81

Figure 4.1. Quantification of extracellular acetate and ethanol during chronological
aging…………………………………………………………………………….............. 93

Figure 4.2. Carbon sources affect chronological aging and stress response..................... 96

Figure 4.3. Technical procedures affected acetic acid accumulation……………........... 99

Figure 4.4. Genetic mutations affecting acetic acid levels, CLS, and pH…….............. 101

Figure 4.5. CR affected ethanol and acetic acid accumulation........................................103

Figure 4.6. Carbon sources and genomic stability...........................................................105

Figure 4.7. Branched chain amino acid leucine affects acetic acid generation…..........106

Figure 4.8. Excessive leucine promoted acetic acid accumulation..................................108

Figure 4.9. Leucine catabolism and acetic acid generation……………………............ 109

Figure 4.10. ACH1 was important for acetic acid utilization………………..…........... 110

Figure 4.11. Respiration and carbon sources utilization………………………............. 112

Figure 4.12. Electron transport activity is important for acetic acid utilization…......... 114

Figure 4.13. Acetic acid and reserve carbon sources.......................................................116

viii

Figure 4.14. A model for the role of the Tor-Sch9 pathway in preventing the switch of
cells to an alternative metabolic mode that promotes longevity…………………......... 118

ix

Abstract
Increased dosage of Sir2, a conserved histone deacetylase, extends replicative life span in
yeast, and possibly worms, and flies and protects mammals against certain diseases.
Previous work in our lab has shown that it is the lack of Sir2 and not its overexpression
that promotes resistance to different stresses and extends chronological lifespan when
combined with calorie restriction (CR) and/or mutations in the Tor/Sch9 or Ras pathways
in yeast. To identify genes and pathways that mimic the effects of CR on aging and
cellular protection we explored the role of Sir2 in regulating stress response and
examined the genetic interaction between Sir2 and stress resistance transcription factors,
Msn2/4 and Gis1. Our results suggest that Msn2/4 and Gis1 are required for the enhanced
stress resistance of sir2 Δ mutant and the further extension of chronological lifespan of
sir2 Δ mutant under caloric restriction. In agreement with this result, the serine/theronine
kinase Rim15, a positive regulator of Msn2/4 and Gis1, was implicated in Sir2 mediated
cellular sensitization to stress. We also examined the role of Sir2 in genomic stability
during chronological aging and proposed a potential mechanism for its action. Further
study of Sir2 implicated its involvement in regulating Ras2 expression. These studies
shed light on the investigation of Sir2's function in higher eukaryotes.

The Sir2 homolog SIRT1 deacetylase, one of the best-characterized sirtuins in mammals,
has been shown to mediate some of the beneficial effects of calorie restriction (CR).
However, its role in CR-dependent lifespan extension still remains unknown and highly
controversial. We previously found that mice lacking both copies of SIRT1 displayed a
x

shorter median lifespan than wild type mice on an ad libitum or a caloric resitriction diet.
Here we demonstrated that the median lifespan of SIRT1+/- heterozygote mice in CR
was identical to that observed in wild type mice but a higher frequency of pathologies
was displayed in SIRT1+/- mice. Microarray gene expression analysis further revealed
the possible relations between SIRT1 and CR. Our results suggest that some SIRT1
expression but not its high expression is required for the beneficial effect of CR in
longevity and health.

In mammals, several days of food deprivation lead to the accumulation of ketone bodies
including acetoacetic acid in the blood. Here we show that as external glucose becomes
depleted, S. cerevisiae convert leucine to the ketone body-like acetic acid, analogously to
the conversion of leucine to acetoacetate in fasting mammals. Acetic acid promoted the
activation of pro-aging pathways similarly to glucose and ethanol. Whereas wild type and
ras2Δ mutant cells accumulated acetic acid, tor1Δ and sch9Δ mutants depleted it rapidly
by a mechanism that required acetate CoA-transferase and that was essential for lifespan
extension. In sch9Δ mutants acetic acid was partly depleted by a mechanism that required
oxidative phosphorylation and was utilized to promote the stress resistance carbon source
trehalose generation. These results indicate that that the effect of Tor/S6K deficiency on
CLS extension involves an alternative metabolic mode, in which acetic acid can be
utilized for energy production and the storage of stress resistance carbon sources. These
effects are reminiscent of those described for mammals in response to fasting and raise
xi

the possibility that the life span extending effect of inhibition of TOR/S6K in higher
eukaryotes may also involve analogous metabolic switches.
1

Chapter 1 Background and Significance
1.1 Yeast model and aging
Aging is a complex process. No living organisms can escape aging and its inevitable
consequence: death. The phenotypes of aging vary from organism to organism, but an
overall physiological decline with age is usually observed. Intensive investigations have
been made for decades to understand the mechanisms of aging. Not surprisingly, the
study of aging is becoming progressively more central in biological and biomedical
research, in part because of the worldwide increase in population and expanded
population of elderly group that is associated with a high rate of costly age-related
diseases, such as cancer. Therefore, it is urgent to understand how aging progresses in
order to develop regimens and pharmaceutical interventions to delay the aging process
and/or to extend health span.

Due to its extreme complexity, the mechanisms of aging are still elusive. Many theories
of aging from evolutionary, molecular, cellular or systemic perspectivehave been
proposed (Weinert and Timiras 2003). Since aging is a multifactorial process, these
theories point to different aspects of aging and may collectively elucidate the aging
mechanisms. Evolutionary and experimental evidences have been accumulated to support
these theories. However, contradictory results are frequently observed in different aging
studies, underlining the need to further explore the mechanisms responsible for
senescence. Because of the difficulty to study aging in humans, genetic model systems
2

are utilized to establish causal relationships between various age-related changes and
aging. In many studies, interventions to extend life span, one of the best ways to identify
genes and pathways that affect aging, have been applied to different model organisms.
Among them the budding yeast, S. cerevisiae, is one of the simplest and best investigated.

The budding yeast Saccharomyces cerevisiae is a unicellular eukaryote with a short life
cycle, which facilitates the study of longevity. In addition, the yeast genome is well
mapped and easy to be manipulated by straightforward molecular and genetic techniques.
There are two major methods to assess yeast aging, chronological and replicative aging.
The replicative life span (RLS) measures the number of daughter cells generated by an
individual mother cell (Mortimer and Johnston 1959; Kaeberlein, McVey et al. 1999).
This assay takes advantage of the asymmetric budding, which facilitates the distinction of
daughter cell from mother cell. The number of divisions is limited. Once cells lose their
dividing ability, they enter senescence and are believed to lose function and die
afterwards. This assay models mitotically active cells in higher organisms. On the other
hand, the chronological life span (CLS) measures long-term survival of yeast cells
maintained in the post-diauxic and stationary phases in the absence of cellular division.

In contrast to the replicative aging assay, CLS assay is able to model the aging of post-
mitotic cells in higher eukaryotes (Longo, Gralla et al. 1996; Fabrizio, Pozza et al. 2001;
MacLean, Harris et al. 2001). Normally, CLS is measured by monitoring the colony
formation of cells after entering the post-diauxic state. The post-diauxic phase and
3

stationary phase are distinct depending on nutrient availability. The post-diauxic phase
begins approximately 24 hours following initial growth of cells in the culture, at the time
when extracellular glucose is depleted, cell growth is greatly reduced and the
mitochondrial respiratory activity is dominated by the catabolism of ethanol produced
from fermentation. Stationary phase is a low metabolism state, which starts at the end of
the post-diauxic phase and up-regulates many stress responsive genes (Werner-
Washburne, Braun et al. 1996; Longo 2003).

The aging regulatory pathways are highly conserved from yeast to mammals (Longo
1999; Longo and Finch 2003; Longo, Mitteldorf et al. 2005). That Saccharomyces
cerevisiae serves as a simple and valuable model for studying aging is partly attributed to
the existence of many homologs and orthologs in higher eukaryotes. In the past, the
budding yeast contributed to the identification of some major conserved or partially
conserved pathways regulating aging, such as the Tor/Sch9 and the Ras/cAMP/PKA
pathways (Beck and Hall 1999; Pedruzzi, Burckert et al. 2000; Fabrizio, Pozza et al. 2001;
Fabrizio, Liou et al. 2003).

1.2 Sir2 and stress response in aging
Silent information regulator 2 (Sir2) is a yeast class III nicotinamide adenine dinucleotide
(NAD)-dependent histone deacetylase, which deacetylates histone H3 and H4 in a NAD
dependent manner(Imai, Armstrong et al. 2000; Landry, Sutton et al. 2000). Sirtuins, the
Sir2 family proteins, are conserved from bacteria to humans (Brachmann, Sherman et al.
4

1995; Frye 2000). Sir2 plays important roles in numerous biological processes, among
which aging is one of the best studied (Kaeberlein, McVey et al. 1999; Fabrizio, Gattazzo
et al. 2005).

The link between Sir2 and aging initially came from yeast. Lack of Sir2 reduced
replicative life span whereas overexpression of one extra copy of Sir2 had the opposite
effect (Kaeberlein, McVey et al. 1999). One proposed mechanism on how Sir2 promoted
replicative life span involves the repression of homologous recombination at rDNA
repeats which in turn inhibits formation of extrachromosomal rDNA circle, a proposed
yeast replicative senescence accelerator (Sinclair and Guarente 1997). Many Sir2
homologs and orthologs were subsequently identified in higher eukaryotes and their roles
in aging investigated. Overexpression of Sir2 orthologs was reported to extend the life
span of C.elegans (Tissenbaum and Guarente 2001) and Drosophila (Rogina and Helfand
2004). In mammals, the Sir2 homolog Sirt1 was suggested to be required for health span
(Boily, Seifert et al. 2008; Finkel, Deng et al. 2009). Sir2, however, has an opposite effect
on chronological aging: lack of Sir2 not only does not decrease but further extends the
chronological lifespan induced by calorie restriction or mutations in the TOR/Sch9, or
Ras/cAMP/PKA pathways in S. cerevisiae (Fabrizio, Gattazzo et al. 2005). Recent study
also challenged life span extension caused by Sir2 in both C. elegans and Drosophila
(Burnett, Valentini et al. 2011).

5

Yeast in its lifecycle encounters various stresses including starvation, UV radiation, heat
and oxidative stress. Among these, oxidative stress has been shown to be tightly related
to the aging progress and oxidative stress resistance is closely linked to lifespan extension
in a variety of organisms (Kregel and Zhang 2007; Romano, Serviddio et al. 2010). The
well-known free radical theory of aging proposes that the free radical-containing reactive
oxygen species (ROS), generated by mitochondrial respiration electron transport chain
leaking, causes cumulative damage and senescence (Harman 1956; Finkel and Holbrook
2000). Supporting evidence for this theory came from the finding of elevated levels of
oxidant-damaged DNA and proteins in aged organisms (Beckman and Ames 1998),as
well as from the study of evolutionary conserved superoxide dismutase (SOD), an
enzyme scavenges superoxide anions. Oxidative stress and damage occur when SOD and
other antioxidant enzymes are not able to process excess ROS. Our lab has shown that
overexpression of the mitochondrial SOD gene SOD2 is sufficient to increase
chronological life span while deletion of its upstream regulators MSN2/4 and RIM15
causes a major decrease in chronological life span (Fabrizio, Liou et al. 2003). Msn2 and
Msn4, two C2H2 zinc finger proteins, bind to the stress response element (STRE) motif
to induce transcription of stress responsive genes (Estruch and Carlson 1993; Martinez-
Pastor, Marchler et al. 1996). The protein kinase Rim15, downstream of Tor/Sch9 and
Ras/cAMP/PKA,regulates both Msn2/4 and Gis1 (Roosen, Engelen et al. 2005; Swinnen,
Wanke et al. 2006; Wei, Fabrizio et al. 2008). Lack of Rim15 shortens the chronological
life span and abrogates cellular protection against oxidative stresses (Fabrizio, Liou et al.
2003; Wei, Fabrizio et al. 2008).
6

In addition to life span extension, lack of Sir2 enhances cellular protection against thermo
and oxidative stresses (Fabrizio, Gattazzo et al. 2005). By contrast, in C.elegans, it was
reported that the yeast Sir2 homolog SIR-2.1 is required for heat shock response (Raynes,
Leckey et al. 2012). In mammals, Sirt1 was also shown to directly or indirectly affecta
large number of non-histone targets, including the stress response forkhead transcription
factors(FOXOs) (Daitoku, Hatta et al. 2004; van der Horst, Tertoolen et al. 2004) and
tumor suppressor p53 (Luo, Nikolaev et al. 2001; Vaziri, Dessain et al. 2001; Langley,
Pearson et al. 2002), both of which are important for oxidative and genotoxic stress
responses. In contrast, our lab found that SirT1 deficiency reduces Ras activation and
ERK1/2 phosphorylation in neurons, which protected neurons against oxidative damage
(Li, Xu et al. 2008).

1.3 Sir2 and genomic instability in aging
Genomic instability, which includes a variety of types of DNA damage, is a hallmark of
aging in eukaryotes. Genomic instability, which is normally associated with accumulation
of mutations and breaks in chromosomes, is proposed to be a major driving force of
tumorigenesis (Shen 2011). Not surprisingly, mutations in DNA repair genes are found in
hereditary cancers (Negrini, Gorgoulis et al. 2010). Various forms of spontaneous
mutations and induced DNA damage accumulate throughout the life time of cells. To
avoid the deleterious consequences of genomic instability, cells employ a variety of
machinery to repair specific types of DNA damage. Single-stranded breaks or lesions and
DNA mismatches are usually repaired by base excision repair (BER), nucleotide excision
7

repair (NER) or translesion DNA synthesis (TLS), and double stranded breaks are
repaired via homologous recombination (HR) and non-homologous end joining (NHEJ)
(Holmquist 1998; Brandsma and Gent 2012; Brierley and Martin 2012; Sharma,
Helchowski et al. 2012). Defects in these repair pathways caused by malfunction of the
repair machinery render cell hypersensitive to UV radiation, alkylation and oxidation,
and lead to aging-like phenotypes, oncogenesis and premature death.

It is well known that the conserved NAD-dependent histone-deacetylase function of Sir2
promotes heterochromatin formation and gene silencing in multiple loci, including the
mating type loci HML or HMR (Rine and Herskowitz 1987), ribosomal DNA (Bryk,
Banerjee et al. 1997; Smith and Boeke 1997; Guarente 1999) and telomeres (Strahl-
Bolsinger, Hecht et al. 1997; Hoppe, Tanny et al. 2002; Xu, Zhang et al. 2007). High
activity of rDNA recombination is hypothesized to be a primary cause of replicative
aging (Lindstrom, Leverich et al. 2011). Sir2 is also suggested to be involved in DNA
double-strand breaks repair by non-homologous end joining (NHEJ) (Lee, Paques et al.
1999). The Sir2 homolog Sirt1 was reported to repress genomic instability by promoting
sufficient DSBs repair through homologous recombination (Oberdoerffer, Michan et al.
2008). Sirt6, another homolog of Sir2, was shown to compromise base excision repair to
promote resistance to DNA damage and prevent age-related damage (Mostoslavsky,
Chua et al. 2006).

8

The role of Sir2 in maintaining genomic stability is still controversial. Previously, our lab
reported that deficiency in the pro-aging protein Sch9 protects against the age-dependent
defects in the RecQ helicase SGS1, homolog of WRN and BLM, by inhibiting the age-
dependent increase in DNA mutations and error-prone recombination (Madia, Gattazzo et
al. 2008). A follow-up study proposed a mechanism that the protection against genomic
instability by deletion of Sch9 was through a Revl/polymerase ζ dependent manner
(Fabrizio, Gattazzo et al. 2005; Madia, Wei et al. 2009). In agreement with our data, the
mammalian Sch9 homolog AKT has been shown to inhibit checkpoint signaling and
repress homologous recombination repair by blocking recruitment of double-strand break
(DSB) machinery, such as RPA, Brca1, and Rad51, to damaged DNA loci, and as a result,
potentially promoting genomic instability and tumorigenesis (Xu, Lao et al. 2012). In
yeast, Sir2 was reported to further decrease the low mutation frequency in Sch9 deficient
cells, which was proposed to be responsible for the additional life span extension. It is
also proposed that translocation of Sir2 in response to DSBs (Martin, Laroche et al. 1999;
Mills, Critcher et al. 1999) allows transient expression of genes in HM loci and
modification of chromatin surrounding the breaks to facilitate repair by recruiting the
repair machinery (Lee, Paques et al. 1999). Alterations in chromatin structure and
consequent gene expression changes are hypothesized to be a force of aging
(Villeponteau 1997; Imai and Kitano 1998). Sirt1 was also shown to alter gene
expression in brain tissues of aged mice (Oberdoerffer, Michan et al. 2008).

9

1.4 Sir2 and caloric restriction in aging
Caloric restriction or dietary restriction is a regimen which involves reduction but not
complete deprivation of nutrients. It is the most robust non-genetic intervention to extend
life span of diverse organisms ranging from yeast (both chronological and replicative) to
mammals (Katewa and Kapahi 2010). Intensive efforts have been made to understand the
mechanism of CR.

In mammals, CR has shown to lower body temperatures and insulin concentrations (Roth,
Lane et al. 2002). Down regulation of the insulin/IGF-1 signaling pathway extends life
span in a wide range of organisms. CR also delays age associated immune decline in
mice (Weindruch, Gottesman et al. 1982). CR was proposed to attenuate oxidative
damage by reducing the rate of metabolism (Weindruch and Walford 1982), although this
is unlikely since a number of studies have shown that CR can even increase metabolic
rates. Based on the free radical theory, less ROS will delay some aspects of the aging
process. Notably, CR has been reported to lower the incidence of aging-related deaths in
rhesus monkeys and delay the onset of age-associated pathologies including diabetes,
cancers, cardiovascular diseases and brain atrophy (Colman, Anderson et al. 2009).
Recently, this discovery was challenged by the primate study performed by another group
who found that reduction in calorie intake in both young and old rhesus monkeys did not
improve survival outcomes (Mattison, Roth et al. 2012). However, other beneficial
effects caused by CR still exist.

10

Initially, the effects of CR on replicative aging were proposed to be mediated by Sir2
(Lin, Defossez et al. 2000). However, this hypothesis was challenged by a subsequent
study showing that CR extended replicative life span in a Sir2 independent manner
(Kaeberlein, Kirkland et al. 2004). One explanation may be provided by the
compensatory effects of other sirtuins, though this possibility it is still under debate
(Lamming, Latorre-Esteves et al. 2005; Kaeberlein, Steffen et al. 2006). Sir2 has also
been suggested to mediate the CR effects in C.elegans and flies (Lin, Defossez et al. 2000;
Rogina and Helfand 2004; Wood, Rogina et al. 2004). In mammals, CR has also been
shown to increase mammalian cell survival in a SIRT1 dependent manner (Cohen, Miller
et al. 2004). However, in yeast chronological aging, CR is still able to prolong life span in
the absence of Sir2 (Fabrizio, Gattazzo et al. 2005). Thus, the role of Sir2 in CR remains
controversial. Thus, more well-designed studies are needed to understand the role of Sir2
in aging and CR-mediated longevity.

1.5 Metabolic regulation in aging
In addition to oxidative stress and genomic instability, metabolic dysregulation is also a
hallmark of aging. Mitochondria are at the center of metabolism and control the
production of energy source ATP and the generation of various cellular metabolites.
Malfunction of mitochondria may result in excessive ROS. Decline of mitochondria
function is associated with many age-related diseases, such as neurodegenerative diseases
(Filosto, Scarpelli et al. 2011). In yeast, lack of mitochondrial respiration severely affects
chronological life span (CLS) (Longo 1999; Aerts, Zabrocki et al. 2009). It has also been
11

shown that caloric restriction increases respiratory activity but decreases mitochondrial
reactive oxygen release (Barros, Bandy et al. 2004).

Glucose metabolism is important for cell survival. Caloric restriction, which lowers
glucose concentration, has been shown to extend yeast replicative and chronological life
span (Lin, Defossez et al. 2000; Wei, Fabrizio et al. 2008). Previously, our lab reported
that depletion of ethanol was sufficient to increase chronological life span (Fabrizio,
Gattazzo et al. 2005). Ethanol is produced from yeast fermentation during growth phase
and is used as carbon source for respiration growth during diauxic shift and postdiauxic
phase (Lillie and Pringle 1980). Glycerol, a byproduct of the glucose metabolism, is
suggested to be a carbon source substitute during caloric restriction. Unlike glucose and
ethanol, glycerol does not adversely affect chronological life span. Instead, its biogenesis
is required for the extended life span caused by Sch9 deficiency (Wei, Fabrizio et al.
2009). Another glucose metabolism by product pyruvate has been implicated in the aging
of mouse oocytes (Liu, Wu et al. 2009). These studies suggest that changes in cell
metabolism and the availability of extracellular carbon sources play an essential role in
aging.

Recently, the yeast chronological aging model driven by conserved effects of pro-aging
pathways and oxidants was challenged by a new mechanism proposed by Burtner et al
(Burtner, Murakami et al. 2009). They defined acetic acid as a cell-extrinsic mediator of
cell death and claimed that dietary restriction or transferring cells to water increased
12

chronological life span by reducing extracellular acetic acid. Furthermore, they claimed
that life span extension by deletion of Sch9 or Ras2 was due to resistance to acetic acid.
Acetic acid is produced from acetaldehyde or acetyl-CoA in glucose fermentation. The
carbon atoms in the acetyl group feeds into the citric acid cycle (Krebs cycle) and is
oxidized for energy production. Acetyl-CoA also provides acetyl group for histone
modification and posttranslational modification acetylation as well. The interchange
between acetic acid and acetyl-CoA may be important for yeast survival. Similarly, when
excessive acetyl-CoA is produced from fatty acid metabolism, ketone bodies are
produced to utilize the extra energy. Acetic acid can induce apoptosis that involves
mitochondria and cytochrome c release (Ludovico, Sousa et al. 2001; Pereira, Chaves et
al. 2010). This acute effect is associated with the dosage of acetic acid that may not
match the physiological level in chronological culture. It is worth mentioning that the
toxic concentration shown by Burtner et al is far higher than the extracellular level
normally measured in culture. IN chapter 4, the notion that acetic acid is at the center of
chronological aging in S. cerevisiaeis clearly challenged by overwhelming evidence
indicating that acetic acid is simply a ketone body-like carbon source which is in fact
important for lifespan extension in sch9 Δ mutants.

13

Chapter 2 Role of Sir2 in Chronological Aging
2.1 Introduction
Silent information regulator 2 (Sir2) is a yeast class III nicotinamide adenine dinucleotide
(NAD)-dependent histone deacetylase, which deacetylates histone H3 and H4 in the
presence of NAD(Imai, Armstrong et al. 2000; Landry, Sutton et al. 2000). The Sir2
family proteinsSirtuins, are conserved from bacteria to humans (Brachmann, Sherman et
al. 1995; Frye 2000). Seven mammalian Sir2 homologs,named SIRT1-SIRT7, have been
identified, which are considered to be functionally related to Sir2 (Frye 2000). Sir2 plays
important roles in numerous biological processes including DNA recombination (Gottlieb
and Esposito 1989; Kaeberlein, McVey et al. 1999), gene silencing (Brachmann,
Sherman et al. 1995), apoptosis (Luo, Nikolaev et al. 2001; Brunet, Sweeney et al. 2004)
and aging (Kaeberlein, McVey et al. 1999; Fabrizio, Gattazzo et al. 2005). In addition to
the histone deacetylase function, sirtuins have been shown to process a large number of
nuclear and cytoplasmic proteins, including the stress response forkhead transcription
factors(FOXOs) (Brunet, Sweeney et al. 2004; Daitoku, Hatta et al. 2004; van der Horst,
Tertoolen et al. 2004), Ku70 (Cohen, Miller et al. 2004), the tumor suppressor p53 (Luo,
Nikolaev et al. 2001; Vaziri, Dessain et al. 2001; Langley, Pearson et al. 2002) and NF-
κB (Yeung, Hoberg et al. 2004).

The role of Sir2 in the aging process and cellular senescence has been intensively
investigated. Sir2 has been proposed to be an anti-aging factor. In yeast, chronological
14

life span (CLS), a measure of chronological survival of a population of cells, and the
replicative life span (RLS), a measure of the maximum number of daughter cells
produced by an individual mother cell, have been the two major methods to assess aging.
Sir2 has been proposed to be a key regulator of replicative life span. Deletion of Sir2
reduces replicative life span while overexpression with one extra copy of Sir2 increases
replicative life span by approximately 35% (Kaeberlein, McVey et al. 1999). The
suggested mechanism of Sir2-mediated replicative longevity is the inhibition of
recombination at rDNA repeats by Sir2 through DNA silencing that subsequently reduces
the accumulation of extrachromosomal rDNA circles, a cell senescence accelerator. Sir2
has also been shown to modify longevity in C.elegans and Drosphila, though through
different mechanisms from those in yeast (Tissenbaum and Guarente 2001; Rogina and
Helfand 2004; Wood, Rogina et al. 2004). Life span extension by increased sir2 in
C.elegans is dependent on the forkhead transcription factor Daf-16, the downstream
effecter of the insulin-like signaling pathway (Kenyon 2001; Tissenbaum and Guarente
2001). In contrast to the anti-aging effect of Sir2 in replicative aging, our lab has shown
that Sir2 plays a pro-aging role in yeast chronological longevity. Lack of Sir2 blocks
further extends the chronological lifespan induced by calorie restriction or mutations in
the TOR/Sch9, or Ras/cAMP/PKA pathways in S. cerevisiae (Fabrizio, Gattazzo et al.
2005). Lack of Sir2 enhanced cellular protection against thermo and oxidative stresses.
Sir2 deficiency also promotes genomic stability and accelerates depletion of extracellular
ethanol, which has been shown to promote chronological aging.

15

Sir2 has been suggested to be a mediator of CR-induced lifespan extension in yeast,
C.elegans and flies (Lin, Defossez et al. 2000; Rogina and Helfand 2004; Wood, Rogina
et al. 2004). However, the role of Sir2 in CR is subject to debate (Lin, Defossez et al.
2000; Kaeberlein, Kirkland et al. 2004; Fabrizio, Gattazzo et al. 2005; Lee, Wilson et al.
2006). CR extends replicative life span in a Sir2 dependent (Lin, Defossez et al. 2000)and
independent manner (Kaeberlein, Kirkland et al. 2004), while in chronological aging, CR
prolongs life span in the absence of Sir2 (Fabrizio, Gattazzo et al. 2005). These
conflicting results provoked studies to understand the role of Sir2 in aging and CR-
mediated longevity.

Accumulating evidences suggest that many genes involved in the regulation of longevity
are evolutionarily conserved in yeast, worms, flies and mice (Longo, Mitteldorf et al.
2005). Activation of the well known conserved glucose or insulin/insulin-like growth
factor-l (IGF-1) signaling pathways, prolongs longevity in organisms raging from yeast to
mammals. In Saccharomyces cerevisiae, mutations in TOR1 or SCH9 (orthologs of
mammalian mTOR and S6K/AKT) or the Ras/cAMP/PKA pathway extend both CLS and
RLS (Fabrizio, Pozza et al. 2001; Kaeberlein, Kirkland et al. 2005), in part by activating
stress resistance serine threonine kinase Rim15 and transcription factors Msn2/4 and Gis1
(Fabrizio, Liou et al. 2003; Wei, Fabrizio et al. 2008).

Msn2 and Msn4 are two closely related C2H2 zinc finger proteins, which were initially
identified as dosage suppressors of the Snf1 protein kinase (Estruch and Carlson 1993).
16

Msn2/4 bind to the STRE (Stress Response Element) motif AGGGG, ARGGGG or G-
RGGGG-GGGG in the promoter region of stress induced genes to induce their
transcription (Estruch and Carlson 1993; Martinez-Pastor, Marchler et al. 1996).The
cytoplasm and nuclear localization of Msn2/4 are regulated by the TORC1 signaling,
which inhibits its translocation to the nucleus (Beck and Hall 1999). In contrast, caloric
restriction positively regulates Msn2/4, promoting its nucleus relocation (Medvedik,
Lamming et al. 2007). Gis1, another C2H2 zinc finger protein, binds to post-diauxic shift
motif (PDS) T(A/T)AGGGAT to activate the transcription of genes with PDS elements in
their promoters (Pedruzzi, Burckert et al. 2000). Both Msn2/4 and Gis1 are regulated by
glucose-repressible protein kinase Rim15, a key effector downstream of Tor/Sch9 and
Ras/cAMP/PKA in response to nutrients (Roosen, Engelen et al. 2005; Swinnen, Wanke
et al. 2006). Our lab has shown that the extended lifespan caused by Sch9 or Ras2
signaling deficiencies is partially mediated by Rim15, Msn2/4 and the downstream
mitochondrial superoxide dismutase (Sod2) (Fabrizio, Liou et al. 2003). Lack of Rim15
abrogates life span extension and diminishes cellular protection against oxidative stresses
(Fabrizio, Liou et al. 2003; Wei, Fabrizio et al. 2008). Furthermore, the CR-mediated
lifespan extension is also dependent on Rim15 and its downstream transcription factors
Msn2/4 and Gis1 (Wei, Fabrizio et al. 2008).

Genomic instability is a hallmark of aging in eukaryotes and is associated with age-
dependent oncogenesis in mammals. Chromosome mutations and breaks accumulate in
response to DNA damage with age. There are also a large number of programmed or
17

spontaneous changes in gene expression, among which some are beneficial but many
could be deleterious to organisms (Vijg 2004; Bahar, Hartmann et al. 2006; Narita,
Krizhanovsky et al. 2006). Previously, our lab reported that the pro-aging factor Sch9, a
homolog of mammalian S6k and Akt, promoted genomic instability and that lack of Sch9
remarkably reduced spontaneous DNA mutations and gross chromosomal rearrangements
(Fabrizio, Gattazzo et al. 2005; Madia, Wei et al. 2009). In mammals, deregulation of
PI3-K/Akt and Ras signaling is frequently associated with oncogenesis. These oncogenes
are implicated to cause tumorigenesis by promoting cellular proliferation and inhibiting
apoptosis of damaged cells (Rodriguez-Viciana, Tetsu et al. 2005; Toker and Yoeli-
Lerner 2006; Yoeli-Lerner and Toker 2006). The conserved NAD-dependent histone-
deacetylase Sir2 promotes heterochromatin formation which in turn silences multiple
genes including genes located in HML or HMR (Rine and Herskowitz 1987) and in
rDNA (Bryk, Banerjee et al. 1997; Smith and Boeke 1997; Guarente 1999). Sir2 was
previously reported to be involved in DNA double-stranded break repair by non-
homologous end joining (NHEJ) (Lee, Paques et al. 1999), although other groups claimed
that the NHEJ function of Sir2 is unimportant for extending life span (Kaeberlein et al.
1999). Recently, Oberdoerffer et al showed that the mammalian sir2 homolog SirT1
promotes DSB repair and suppressed age-dependent gene expression changes
(Oberdoerffer, Michan et al. 2008).

In our study, we explored the role that Sir2 plays in chronological aging and explored the
underlying mechanisms through which Sir2 inhibits stress response and promotes
18

genomic instability in S. cerevisiae during aging. Our data suggest that the enhanced
resistance to oxidative stress and lifespan extension under CR in Sir2 deficient strain is
partially mediated by Msn2/4 and Gis1. Lack of Sir2 greatly evokes homologous
recombination events, which may enhance error free double strand break recombination.
We also elucidated the potential involvement of Sir2 in regulating Ras2 signaling. In
contrast to the anti-aging effects of Sir2, our findings further support a pro-aging role of
Sir2 in yeast and shed light on the study of Sir2 homologs in various cellular processes in
mammals including aging and age related diseases.

2.2 Materials and Methods
Yeast strains Majority of the Saccharomyces cerevisiae strains used in this study were
derived from DBY746 (MATα, leu2-3, 112, his3∆1, trp1-289, ura3-52, GAL+). Some
experiments were also performed in W303AR (MATa, leu2-3, 112, trp1-1, ura3-52, his3-
11) and the derivatives to confirm the results obtained with DBY746 strains. One-step
gene replacement using plasmid pJH103.1 (provided by D. Moadez, Harvard University)
was used to disrupt SIR2 gene. Strains expressing Sir2 protein was obtained by
transforming sir2 Δ mutants with a centomeric plasmid carrying wild-type SIR2 (p-SIR2-
LEU2) (provided by D. Moadez, Harvard University). Knockout strains were generated
by one-step gene replacement as described previously (Brachmann, Davies et al. 1998).
The sch9 ∆, cyr1::mTn mutants (Fabrizio, Pozza et al. 2001)and ras2 Δ mutant (Longo
1996) have been described previously. Strains with STRE-lacZ reporter gene were
19

generated by transforming the plasmid pMM2 containing four tandem repeats of STRE
motif from the HSP12 sequence (-221 to -241) (Boy-Marcotte, Perrot et al. 1998) and
integrating into the URA3 locus of wild type. The plasmid pCDV454 containing LacZ
reporter under the control of a 37bp SSA3-PDS region (-206 to -170) was integrated into
the URA3 locus of wild-type cells to generate PDS-lacZ reporter gene strain (Pedruzzi,
Burckert et al. 2000). The reporter gene activities have been described in previous study
(Wei, Fabrizio et al. 2008). Strains overexpressing Gis1 protein was generated by
transforming with YCpADH1-GIS1 plasmid (a gift from Dr. Claudio De Virgilio,
Botanisches Institut der Universität) (Pedruzzi, Burckert et al. 2000) and with Msn2-GFP
fusion protein expression were generated by transforming with pMsn2-GFP plasmid
(Gorner, Durchschlag et al. 1998) as described previously.

Growth conditions Yeast cells were grown in SDC containing 2% or 0.5% glucose
supplemented with a 4-fold excess of the three essential amino acids tryptophan, leucine,
histidine and uracil to avoid possible lifespan modification due to auxotrophic
deficiencies of the strains.

Chronological life span assay Yeast chronological life span was measured as previously
described (Fabrizio, Pozza et al. 2001). Overnight SDC culture was diluted (OD0.1) in to
fresh SDC medium to a final volume of 10 ml (with 5:1 flask to culture volume) and
were maintained at 30°C with shaking (200 rpm).Twenty four hours later was considered
as day1. Every 48 hours, properly diluted culture was plated on to YPD plates and then
20

incubated at 30°C for 2-3 days. Chronological life span was monitored by measuring
colony-forming units (CFUs). Day3 CFUs was considered as the initial survival (100%)
and used to determine the age-dependent mortality. For medium switch experiment, wild
type and sir2 Δ mutant cells were grown in SDC for 3 days, centrifuged to obtain the
supernatant (expired medium), washed twice with sterile distilled water, and suspended
in expired medium from each other. For extreme CR experiment, cells were grown in
SDC medium till day1 or day3, centrifuged and washed twice with sterile nanopure water,
then added with sterile water. The same procedure was performed every other or four day.

In situ viability assay Day 1 or day 3 SDC cultures of tryptophan auxotrophic strains
were diluted and plated on to SC-Trp plates (~200 cells/plate) with 2% glucose, ethanol
or acetic acid. Plates were incubated at 30°C for the duration of the experiment. Half
milliliter of 2 mg/ml tryptophan was added to –TPR plates. After 2-3 days incubation at
30°C, colony formation was recorded. Details and discussion about this assay is
described by Wei et al (Wei, Madia et al. 2011).

Stress resistance assay Heat shock resistance was measured by spotting serial dilutions
(5-fold or 10-fold dilution) of cells removed from cell cultures onto YPD plates and
incubating at 55°C (heat-shocked) and at 30°C (control) for 60–150 min. After the heat
shock, plates were transferred to 30°C and incubated for 2 days. For oxidative stress-
resistance assays, cells were diluted 10 fold in K-phosphate buffer, pH6.0, and treated
21

with 70- 300mM H2O2 for 30 min. Serial dilutions (5fold or 10 fold) of untreated control
and H2O2-treated cells were spotted onto YPD plates and incubated at 30°C for 2–3 days.

LacZ reporter gene assay 1mL culture was collected and washed once with water and
then stored at -80 °C. Add 100ul low-salt lysis buffer(50 mM Tris, pH7.5, 0.1%(v/v)
Triton X-100, 1X protease inhibitor,100 mM NaCl, 2.0 mM EDTA, 2.0 mM EGTA,50
mM NaF) and equal volume glass beads to the cell pellets. Vortex at max speed for 45s,
chill on ice for 45s, repeat 5 times. Centrifuge at 14,000 rpm at 4 °C for 5min and transfer
the supernatant to a clean tube. The protein concentration of the lysate was assayed with a
BCA kit (Pierce).

Mix 55 ul of lysate (or appropriately diluted samples) with 85 μL of Substrate solution
(1.1 mg/ml ONPG in Z Buffer: 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1
mM MgSO4, 50 mM 2-mercaptoethanol, pH7.0). Read Absorbance at 420 nm every 5
min until 30 min after the initiation of reaction. LacZ activities were determined by
fitting the A420/time data to that of serial diluted recombinant Beta-galactosidase
(Promega) and were further normalized with total protein.

Mutation frequency assay A wide spectrum of spontaneous mutation frequency was
monitored by measuring the CAN1 (YEL063) gene mutation frequency(Chen, Umezu et
al. 1998; Madia, Gattazzo et al. 2007). The CAN1 gene, encoding arginine permease can
also uptake the arginine analog canavanine, which is toxic to the cells at the
22

concentration of 60 mg/L. Overnight culture were diluted in SDC medium to OD600 0.1.
Cell viability was described in Chronological life span assay. To monitor canavanine-
resistant mutants, an appropriate volume of culture was collected (normally, the collected
culture contains 2X10
7
cells) starting at day1 then every other day. The culture was
centrifuged and washed once with sterile water and plated onto selective medium (SDC
minus arginine supplemented with 60 ug/ml L- canavanine sulfate). Mutant colonies were
counted after 3 – 4 days incubation at 30
o
C. The ratio of canavanine-resistant mutants
over total viable cells was denoted as mutation frequency.

A DBY476 background strain with URA3 cassette replaced HXT13 (YEL069), which
encodes for a highly redundant hexose transporter, was generated to detect Gross
chromosomal rearrangements (GCRs) (Chen and Kolodner 1999; Madia, Gattazzo et al.
2007). Both HXT13 and CAN1 are located on chromosome V, 7.5kb apart. Cells obtain
canavanine and 5-fluorotic acid (5FOA) resistance when lossing both CAN1 and URA3.
This experiment was operated similarly to CAN1 gene mutation frequency assay as
described above. Since the GCRs frequency is relative low compared to CAN1 gene
mutation frequency, a larger volume of culture (contains 2-3X10^8 cells) are needed for
plating. The medium for detecting GCRs are SDC minus arginine supplemented with 1
mg/ml 5FOA and 60ug/ml L- canavanine. Colony units are counted after 3-4 days
incubated at 30
o
C. The ratio of mutants vs viable cells was described as GCRs frequency.

23

For detecting the frequency of a base substitution, we measured the frequency of trp- to
Trp + reversions (Capizzi and Jameson 1973; Madia, Gattazzo et al. 2007) in DBY746
strains which carry a trp1-289 amber mutation (C → T at residue 403 of the coding
sequence). The experiment procedure is also similar to CAN1 mutation frequency assay.
Around 10^8 cells are needed to platet onto selective medium SDC –TPR in order to
observe of fair amount of reversion events. The reversion frequency is described as the
ratio of revertants over viable cells.

Homologous and homeologous recombination assay To monitor the homologous
(100%) and homeologous (91%) recombination events, we generated strains integrated
with linearized plasmids carrying either 100% homologous HIS3::intron-IR (pRS406) or
91% homeologous IRs (pRS407) as describe previously (Datta, Adjiri et al. 1996).
Chronological aging culture which contains about 2 × 10
7
viable cells was collected
starting from day 1, washed once with sterile water and plated onto selective medium
SDC-HIS supplemented with galactose according to Datta et al (Datta, Adjiri et al. 1996)
every other day. Homologous or homeologous His
+
recombinants were measured by
counting the colony formation units after 3-4 days incubation at 30
o
C. The frequency of
homologous or homeologous recombination was evaluated by the ratio of recombinants
over viable cells.

Quantification of mRNA by real-time PCR Total mRNA was extracted from cells in
expired cultures with a standard phenol/chloroform method. In brief, 2-3 ml culture was
24

collected, centrifuged and washed twice with sterile water. The cell pellets were added
with 400 ul TES (10 mM Tris, pH7.5, 10 mM EDTA, 0.5% SDS) buffer and 400ul acidic
phenol on ice and vortexed for 15s. Then the mix was incubated at 65-70°C for 20min,
vortex for 10s every 5min and afterwards incubated on ice for 5’, centrifuged for 5’ at
4°C. Transfer the top layer to a new tube and extract once with 400ul CHCl3. The
extracted top layer was added with 1 volume 3 M NaOAc (pH5.2) and 3 volume of
ethanol and stored at -80°C for 30min. Then centrifuge the mix for 15min at max speed at
4°C, briefly dry and resuspend the extracted RNA in DEPC water.RNA was reverse-
transcribed using RetroScript II reverse transcription (Invitrogen) and random primers.
Real-time PCR was performed using the DNA Engine Opticon 2 (BioRad) with YBR-
green I dye (Bio-Rad laboratory).Gene expression levels were normalized to
housekeeping gene ACT1, encoding for actin and expressed as the ratio to wild type.

Western blotting and immunoprecipitation (IP) Cells were collected and centrifuged
at 13X1000 rpm for 1min. The cell pellet was washed once with water (can be saved at -
80 degree for future use) and added with low-salt lysis buffer (50mM Tris, PH7.5,
0.1%(v/v) Triton X-100 or NP40, 1Xprotease inhibitor (cocktail, sigma P2714), 100mM
NaCl, 2.0mM EDTA,2.0mM EGTA,50mM NaF, 10% glycerol) and equal volume glass
beads (Sigma G8772). The cells were broken by violent vortex (highest speed) 45s for 5-
6 times with 45s stop each repeat. The total protein is obtained by centrifuging the lysate
and collecting the supernatant. The protein concentration is decided by using BCA
protein assay kit. Equal amounts of protein were treated with Laemmli buffer and boiling
25

at 95°C for 5min. The boiled protein samples were resolved on SDS-PAGE and
immunoblotted with indicated antibodies for ECL detection. The blots were quantified
using ImageJ and band intensities were normalized to GAPDH. Antibodies against
SIRT1 (07-131), Anti-Ras, clone RAS10 (05-516) were obtained from Upstate. Anti-
GAPDH (ab9484) antibody was from Abcam (Cambridge, MA). Anti-acetylated-lysine
(Ac-K-103) (9681) was obtained from Sigma. Anti-Myc antibody (R950-25) was
obtained from life technologies.

For immunoprecipitation, cells were lysed in low-salt buffer as described above. Equal
amount of protein was added with A/G-Sepharose beads and indicated antibodies and
incubated at 4
o
C with agitation for 2 hours or overnight. The mix was washed with bead
buffer (50mM NaF, 50mM Tris, PH7.4, 5.0mM EGTA, 5.0mM EDTA, 250mM NaCl,
0.1% NP-40) for three times. The beads were added with 2X protein sample buffer (0.002%
(w/v) bromophenol blue, 40% (v/v) Glycerol, 4% (w/v) 2-mercaptoethanol or 6.2% (w/v)
DTT, 250mM Tris) and boiling at 95°C for 5min. Centrifuge the boiled sample and add
equal volume into SDS-PAGE gel and immunoblotted with indicated antibodies for ECL
detection.

GFP fluorescence microscopy Aliquots (100ul) of the cultures from different strains
were washed with sterile water and added with 20ul water. The enriched cells were put
on microscope slides and covered with 18 × 18 mm cover slides. Cells were viewed with
a Leica fluorescence microscope and images were captured.
26

Statistical analysis Longevity curves and mutation-frequency bar graphs were analyzed
by two tailed t test (P < 0.05). Procedures for microarray data analysis based on Gene
Ontology have been described previously (Cheng, Fabrizio et al. 2007).

2.3 Results
2.3.1 Lack of Sir2 promotes stress resistance through Gis1 and Msn2/4
Previously, our lab has shown that Sir2 blocks extreme chronological lifespan caused by
calorie restriction or mutations in the Tor/Sch9, or Ras/cAMP/PKA pathways in S.
cerevisiae (Figure 2.1C) (Fabrizio, Gattazzo et al. 2005). However, the underlying
mechanism of how Sir2 regulates chronological aging remains elusive. My dissertation
will describe the effort we made to understand the regulatory role of Sir2 in aging.

27

A B

C D

Figure 2.1 Sir2 blocks extreme life span extension and reduces stress resistance in a
medium independent manner. (A) Day3 yeast cells were heat-shocked at 55°C or treated
with 100 mM H2O2 for 30 min or 100uM menadione for 1hr, serially diluted, and
spotted onto YPD plates. Strains shown are sir2 Δ mutant, and sir2 Δ mutant transformed
with centromeric plasmid pSIR2, which carries the SIR2 wild-type gene, and plasmids
pSIR2-H364Y and pSIR2-G262A, which carry Sir2 deacetyalse-defective enzymes
alleles; sch9 Δ, sch9 Δsir2 Δ and sch9 Δsir2 Δhmra Δ mutants. Data are adapted from
Fabrizio et al, 2005. (C) Chronological life span (CLS) of sir2 Δ, sch9 Δ, sch9 Δsir2 Δ
mutants in SDC Medium. (D) Wild type and sir2 Δ mutant cells grown in SDC medium
supplemented with 2% glucose. On day3, media from the two strains were exchanged.
Then cells were exposed to heat stress (55
o
C) or to H2O2 (150 mM) for 30min. WT swi
and sir2 swi indicates that cells were transferred to the different medium.

1 3 5 7 9 1113151719212325272931333537394143
0
25
50
75
100
125
WT(DBY746)
sir2Δ
sch9Δ
sch9Δsir2Δ
day
Survival (%)
28

Lack of Sir2 has been shown to greatly promote cellular protection against thermo and
oxidative stresses. This effect is independent of the mating type desilencing (Figure 2.1A,
B). To exclude the possibility that the enhanced stress response is medium-dependent, we
switched the media between wild type and sir2 Δ mutants. Day3 wild type expired
medium was added to sir2 Δ mutant cells and day3 sir2 Δ mutant expired medium was
added to wild type cells. Two hours after the switch, the same procedure was performed
again to minimize the changes in the medium. Two hour later, cells were exposed to heat-
shock (55°C) for 90 min or 2 hrs and hydrogen peroxide (150mM) for 30min (Figure
2.1D). The sir2 Δ mutant still show strong stress resistance compared to wild type in both
wild type and its own media, which indicated the enhanced stress resistances of sir2 Δ
mutants are independent of medium components.

Since in Sir2 deficient cells many stress response genes are up-regulated, we
hypothesized that Sir2 regulates stress responsive transcription factors, which control the
expression level of stress response genes. Previously, Fabrizio et al showed that extended
chronological survival in ras2 Δ and cyr1:Tn mutants was associated with stress resistance
and required stress-resistance transcription factors Msn2/Msn4 and Mn-superoxide
dismutase (Sod2) (Fabrizio, Liou et al. 2003).

29

A

C

D E

Figure 2.2 Thermal and Oxidative Stress Resistance and chronological life span (CLS) of
cells deficient in Sir2 and stress response transcription regulators Msn2, Msn4 and/or
Gis1 and protein kinase Rim15. Chronological aging day 3 cells were exposed to heat
stress (55
o
C) or to H2O2 (100 mM) for 30min. (A) Strains shown are wild type
(DBY746), sir2 Δ, gis1 Δ, gis1 Δsir2 Δ, msn2/4 Δ and msn2/4 Δsir2 Δ. (B) Wild type
(DBY746), sir2 Δ, msn2/4 Δ gis1Δ and msn2/4Δ gis1Δ sir2 Δ. (C) Wild type (DBY746),
sir2 Δ, rim15 Δ and sir21 Δrim15 Δ. (D) CLS of wild type (DBY746), sir2 Δ, gis1 Δ,
gis1Δsir2 Δ, msn2/4 Δ and msn2/4Δsir2 Δ. (E) CLS of wild type (DBY746), sir2 Δ,
msn2/4Δ gis1 Δ and msn2/4 Δ gis1 Δ sir2 Δ, rim15 Δ and sir21 Δrim15 Δ.

To test if the enhanced stress resistance of Sir2 deficient cell is dependent on the three
stress response transcription factors Msn2/4, Gis1, we deleted Msn2/4, Gis1 or both in
0 2 4 6 8 10 12 14
0
25
50
75
100
125
WT(DBY746)
sir2Δ
gis1Δ
sir2Δgis1Δ
msn2/4Δ
msn2/4Δsir2Δ
day
Survival (%)
1 3 5 7 9 11 13 15 17
0
25
50
75
100
125
WT(DBY746)
sir2Δ
msn2/4Δgis1Δ
msn2/4Δgis1Δsir2Δ
rim15Δ
rim15Δsir2Δ
day
Survival (%)
30

sir2 Δ mutant. Day3 chronological aging cells of the double, triple or quadruple deletion
mutants were treated with heat-shock (55
o
C) for 60-100 min and H2O2 (100mM) for
30min (Figure 2.2A, B). Lack of Msn2/4 dramatically reduced the thermo resistance of
sir2 Δ mutants and almost reversed the resistance to oxidative stress. Deletion of Gis1 also
reversed the resistance of sir2 Δ mutants to oxidative stress but had a small effect on
thermo stress resistance. The quadruple msn2/4Δgis1Δsir2 Δ mutant was hypersensitive to
oxidative stress but partially maintained the resistance to thermo stress. Therefore, the
enhanced cellular protection against oxidative stress by lack of Sir2 is mediated by
Msn2/4 and Gis1.

To further elucidate the role of stress response transcription factors in the enhanced stress
resistance caused by the deletion of Sir2, we deleted the glucose-repressible protein
kinase Rim15, which regulates Msn2/4 and Gis1 activity. Similar to msn2/4 Δgis1Δsir2 Δ,
deletion of Rim15 in sir2 Δ mutants abolished the increased oxidative stress resistance
and still partially retained the resistance to thermo stress (Figure 2.2C). We also asked if
Msn2/4, Gis1 and Rim15 affected the chronological survival of sir2 Δ mutants. The mean
lifespan of msn2/4Δ, gis1 Δ and rim15 Δ were slightly reduced compared to wild type and
lack of these regulators reduced the life span of sir2 Δ mutants to the same extent as that
in wild type (Figure 2.2 D, E).
31

Figure 2.3 Sir2 reduces the expression of genes with PDS element in their promoter
regions. Yeast cells integrated with STRE- or PDS-lacZ gene elements were grown in
SDC medium. The STRE-lacZ (A, C) and PDS-lacZ (B, D, E) activities were measured
on Day1, 3, 5 or 7. Data shown are means ± SEM (n = 3–6) except sch9 Δsir2 Δ mutants.

To exam the specificity of stress response factors regulated by Sir2, we employed the
STRE-and PDS-driven LacZ reporter gene assay to examine the gene expression in sir2 Δ
mutants. Strains carrying either STRE- or PDS- driven lacZ reporter gene grew in SDC
medium and were collected every other day. Lack of Sir2 did not result in significant
1 3 5 7
0.0
0.2
0.4
0.6
0.8
WT
sir2
sir2 pSIR2
Day
PDS LacZ Activity
u/mg
1 3 5 7
0.0
0.5
1.0
1.5
2.0
2.5
Day
STRE LacZ Activity
u/mg
1 3 5
0
1
2
3
4
WT(DBY746)
gis1Δ
msn2/4Δ
GIS1ox
Day
STRE LacZ Activity
u/mg
1 3 5
0.0
0.2
0.4
0.6
0.8
WT(DBY746)
gis1Δ
GIS1ox
msn4Δ
msn2Δ

Day
PDS LacZ Activity
u/mg
C D
AB
1 3 5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
WT(DBY746)
sir2Δ
sch9Δ
sch9Δsir2Δ

E
day
PDS LacZ Activity
u/mg
32

increase of STRE-dependent transcription during chronological aging compared to wild
type (Figure 2.3A). However, the sir2 Δ mutants transformed with the centromeric
plasmid pSIR2, which carries the SIR2 wild-type gene, significantly reduced the STRE-
driven LacZ activity, indicating the regulatory role of Sir2 in STRE-driven gene
expression (Figure 2.3A). A dramatic increase in PDS-dependent transactivation was
observed on day1 and day3 in sir2 Δ mutants (Figure 2.3B). Lack of Sir2 caused almost
four times increase in PDS-dependent transcription on day1 and two times increase on
day3. At days 5 and 7, no significant difference was observed between wild type and
sir2 Δ mutants. Overexpression Sir2 in sir2 Δ mutants decreased the PDS-driven
transcription to the level in wild type, which suggests Sir2 is a strong regulator of PDS-
driven gene expression in early survival. The STRE element containing genes are majorly
regulated by Msn2/4 and PDS element containing genes that are under the control of Gis1
especially during aging. This was further confirmed by our LacZ reporter gene assay.
Deletion of MSN2and MSN4 greatly inhibited the STRE-driven LacZ transactivation
activity (Figure 2.3C) and lack of Gis1 highly reduced the PDS-driven LacZ
transactivation (Figure 2.3D). To our surprise, deletion of GIS1 enhanced STRE-driven
LacZ activity (Figure 2.3C), suggesting that Gis1 inhibits STRE-driven gene expressions.
In agreement with our stress resistance data shown above, the compensatory activation of
STRE-driven transcription explains the residual stress resistance of gis1 Δsir2 Δ double
mutants.

33

A

B

C D

E

Figure 2.4 Thermal and oxidative stress resistance of cells deficient in Sch9, Sir2, Gis1
and protein kinase Rim15. Chronological aging day 3 cells were exposed to heat stress
(55
o
C) or to H2O2 (150mM) for 30min. (A) Strains shown are wild type (DBY746),
sch9 Δsir2 Δ, sch9 Δgis1 Δ, sch9 Δsir2 Δgis1Δ (B) sch9 Δ, sch9 Δrim15 Δ, sch9 Δsir2 Δ,
sch9 Δsir2 Δrim15 Δ. (C) CLS of sch9 Δ, sch9 Δsir2 Δ, sch9 Δgis1Δ, sch9 Δsir2 Δgis1Δ. (D)
Chronological lifespan (CLS) of cells under extreme CR (label as eCR), where day 3
SDC cultures were washed and switched to water.
0 2 4 6 8 10 12 14 16
0
25
50
75
100
125
sch9Δ
sch9Δsir2Δ
sch9Δgis1Δsir2Δ
sch9Δgis1Δ
day
Survival (%)
0 2 4 6 8 10 12 14 16
0
25
50
75
100
125
sch9Δ
sch9Δsir2Δ
sch9Δrim15Δsir2Δ
sch9Δrim15Δ
day
Survival (%)
3 5 7 9 11131517192123252729313335
0
50
100
150
WT
sir2
WT (eCR)
sir2 (eCR)
msn2/4 gis1 (eCR)
msn2/4 gis1 sir2 (eCR)
rim15 (eCR)
rim15 sir2 (eCR)
Day
Survival (%)
34

Based on the lacZ-reporter gene assay, we concluded that Sir2 inhibits stress response
through the stress response transcription factor Gis1. Therefore, we ask if Gis1 also
mediate the further life span extension of sch9 Δ mutants by deletion of Sir2. Deficiency
in Sch9 greatly enhanced the PDS-driven transactivation, which is further increased by
deletion of Sir2 (Figure 2.3E). This suggests that there are additive regulation of Gis1 by
Sch9 and Sir2. Deficiency in Gis1 almost completely reversed the life span extension by
deletion of Sch9 (Figure 2.4C). Deletion of Sir2 in sch9 Δgis1Δ mutant did not further
improve the chronological survival, which supported our hypothesis that the blockage of
extreme life span by Sir2 deletion was dependent on Gis1. Consistent with the results of
stress resistance assay, removal of Gis1 did not impair the enhanced resistance to thermo
and oxidative stress of sch9 Δorsch9 Δsir2 Δ mutants (Figure 2.4A).

We also looked at the role of the upstream regulator Rim15 in the chronological
longevity. In agreement with the results by Wei et al, lack of Rim15 abolished both the
life span extension and resistance to oxidative stress of sch9 Δ and sch9 Δsir2 Δ mutants
(Figure 2.4B, D) but not the thermo stress resistance, which implied additional
downstream effectors might contribute to the thermo stress response. These observations
confirmed the significance of Gis1 in mediating life span extension of cells lack of Sch9
and/or Sir2. Thus, lack of Rim15 diminished the activity of Gis1 and Msn2/4, which
caused the inefficient cellular protection to oxidative stress of sch9 Δ and sch9 Δsir2 Δ
mutants.

35

Fabrizio et al uncovered that Sir2 blocks the life span extension by caloric extension and
Wei et al elucidated that life span extension by caloric extension depends on Rim15 and
downstream transcription factors Msn2/4 and Gis1 (Fabrizio, Gattazzo et al. 2005; Wei,
Fabrizio et al. 2008). We asked if Msn2/4 and Gis1 mediated the life span extension of
sir2 Δ mutants under caloric restriction. Cells grown in SDC medium were switched to
sterile water on day3. This extreme caloric restriction condition mimics the extreme
starvation condition encountered by yeast in wild. This severe CR condition causes entry
of yeast cells into a low metabolic phase. Under the extreme CR condition, mean life
span of the msn2 Δmsn4 Δgis1Δ mutant slightly shorter than wild type while about 30%
reduction in maximum life span (the age of 10% of the cells alive) was observed. The
quadruple msn2 Δmsn4 Δgis1Δsir2 Δ mutant had similar mean life span but shorter
maximum life span compared to the msn2 Δmsn4 Δgis1Δ mutant. This suggests that both
Msn2/4 and Gis1 mediates the life span extension of Sir2 Δ mutants under caloric
restriction. In contrast, extreme CR failed to extend the chronological survival of Rim15-
null mutant (Figure 2.4E). Deletion of Rim15 in sir2 deficient cell significantly reduced
both the mean and maximum life span but had a 20% increase in mean life span in
contrast with rim15 Δ single mutants (Figure 2.4E), suggesting that additional factors may
contribute to the life span extension of cells lack of Sir2 under extreme caloric restriction.
36

Figure 2.5 Gis1 over expression doesn’t increase life span. CLS of WT (DBY746), sir2 Δ
and sch9 Δ mutants with Gis1 overexpressor.

As Gis1 is essential for the life span extension by deletion of Sch9 and Sir2, we
hypothesized that overexpression of Gis1 could increase chronological longevity.
Therefore we transformed wild type, sch9 Δ and sir2 Δ mutants with Gis1 expression
plasmid and monitored the chronological viability (Figure 2.5). To our surprise, the over
expression of Gis1 did not promote longevity instead it shortened the life span of wild
type and sch9 Δ mutants but not sir2 Δmutants.Thus, high level of Gis1 in the absence of
other cellular changes is not beneficial for chronological survival. This suggested that the
transcription activity mediated by Gis1 is under strict regulation. Since Gis1 serves as
both transcription activator and repressor, the level of genes should be accurately
regulated to maximize chronological life span. The over expression of Gis1 may affect
this balance and be deleterious to survival.

1 3 5 7 9 11 13 15
0
50
100
150
WT(DBY746)
sir2Δ
sch9Δ
GIS1 ox
sir2Δ GIS1 ox
sch9Δ Gis1 ox
day
Survival (%)

A

C
F
fr
ow
m
re
fo
C
p
fr
ad

A
C
igure 2.6 Si
rom wild-typ
wn promote
mutants with
eversible, an
our hours af
Cell lyastes
erformed wi
rom cell wit
dded but und

ir2 reduces
pe and sir2 Δ
er) on day1
h myc-tagge
nd cell-perm
fter treatmen
were immu
ith anti-Myc
thout Myc-ta
derwent all i

the fragmen
Δ mutant ce
, 3, 5, 7, u
d Gis1 wer
eable protea
nt. (C) wild-
unoprecipite
c antibody o
agged Gis1
immunoprec

ntation of G
ells with My
using anti-M
re treated w
asome inhibi
-type and sir
edwith anti-
or anti-acetyl
and blank m
cipitation pro
B

Gis1 protein.
yc-tagged G
Myc antibody
with MG132
itor. Western
r2 Δ mutant
-myc antibo
lated lysine
means sampl
ocedure.
(A) Wester
Gis1 (under t
y. (B) Wild
(50uM), a
n Blot was p
cellswere co
ody and we
antibody. C
le without an

rn blot of ly
the control o
d-type and s
specific, po
performed tw
ollected on d
estern blot
Ctr means sa
nti-myc anti
37
ysates
of its
sir2 Δ
otent,
wenty
day3.
were
ample
ibody
38

Our results have demonstrated that Gis1 mediated the further life span extension by
deletion of Sir2 in long-lived mutants. But the mechanism by which Sir2 regulates Gis1
is unknown. The microarray gene expression file did not reveal increase at mRNA level
for GIS1, MSN2 or MSN4, which indicated that Sir2 probably did not inhibit Gis1 activity
at transcription level. As I mentioned earlier, the mammalian Sir2 homolog SIRT1 has
been shown to regulate several stress-responsive factors, such as forkhead transcription
factor (FOXO1), p53 tumor suppressor, and NF- κB at posttranslational level through
direct protein interaction and modification (deacetylation). Therefore, we proposed that
Sir2 regulated Gis1 at posttranslational level by its deacetylation activity. To test this
hypothesis, we first tagged the chromosomal GIS1 open reading frame, which was under
the control of its native promoter, with a Myc Tag, a polypeptide protein tag derived from
the c-myc gene, at the C-terminus in wild type and sir2 Δ mutants. Western blot was
performed to examine the protein level during chronological aging in both wild type and
sir2 Δ mutant. To our surprise, besides a full length Gis1 ORF with the expected size of
120kDa (the GIS1 ORF (894aa, ~100kDa) plus the myc tag (~20kDa)), there were
multiple variants of the Gis1 protein observed in chronological aging cells (Figure 2.6A).
One variant was about 100kDa and the other four variants were much smaller than the
full length Gis1. The level of the complete Gis1 protein increased with age and the
smaller variants maintained at relatively constant level through the whole life in wild type
cells. Unexpectedly, a lower level of full length of Gis1 and higher level of the small
variants were seen in Sir2 deficient cells. Initially, we considered the multiple variants of
Gis1 might be caused by the myc tag. Recently, Zhang et al published that the
39

transcription activity of Gis1 is negatively regulated by proteasome-mediated proteolysis
(Zhang and Oliver 2010). They also tagged Gis1 with myc tag and the western blot
showed similar pattern of protein variants as ours. The consistency between our data
proved the authenticity of our results. It seemed that the decreased level of full length
Gis1 in sir2 Δ mutants did not correlate with the up-regulated Gis1 activity implied by the
LacZ reporter gene assay. However, Gis1 over-expression showed cellular toxicity and
Zhang et al also reported that the level of the small protein variants increased upon
rapamycin treatment, which suggests down regulation of TOR signaling pathway
promotes proteasome mediated proteolysis of Gis1. This process is possibly beneficial to
cell survival and explains enhanced stress response and improved cell survival of sir2 Δ
mutants.

To examine if Sir2 also regulates the proteasome-mediated Gis1 proteolysis, we treated
pdr5 Δ and sir2 Δpdr5 Δ Gis1-Myc tagged mutant cells with MG132, a specific, potent,
reversible, and cell-permeable proteasome inhibitor. MG132 reduces the degradation of
ubiquitin-conjugated proteins by the 26S complex without affecting its ATPase or
isopeptidase activities. Deletion of PDR5 renders cells permeable to MG132(Zhang and
Oliver 2010). The treatment of MG132 slightly increased the full length Gis1 in both
pdr5 Δ and sir2 Δpdr5 Δ mutants with lower level of complete Gis1 in sir2 Δpdr5 Δmutants
(Figure 2.6B). This implied Sir2 inhibited the degradation of Gis1 protein and this
inhibition was probably by posttranslational modification. Therefore we performed
immunoprecipitation to test if Gis1 was modified by acetylation. The cell lysates from
40

cells with myc tagged Gis1 were precipitated with anti-myc antibody and western blot
was employed to detect the immune precipitates using anti-myc and anti-acetylated lysine
antibody. Unfortunately, we did not find any acetylation modification of Gis1 protein
after making great effort to improve the condition for immunoprecipitation (Figure 2.6C).
One of the intrinsic difficulty was that there were many Gis1 protein variants and the
level of full length Gis1 was relatively low even we enriched the lysates by increasing the
cell volumes. The long immunoprecipitation process may enhance the degradation of
Gis1 and minimize the chance to detect any posttranslational modification. Thus, Gis1-
Myc overexpressor may be a better choice to avoid these technical problems.

Figure 2.7 Sir2 does not inhibit Msn2 nucleus localization. Wild type and sir2 Δ
mutantswith GFP-tagged Msn2 expressing plasmid were visualized under fluorescent
microscope in growth phase and on day1 and day3.

41

So far, by what means Sir2 regulated Gis1 activity was still unclear. Both Gis1 gene
expression file and posttranslational modification should be examined by RT-PCR and
using Gis1 overexpressor. Our results suggested that Gis1 played important role in
promoted longevity by blocking Sir2 activity.

Although lack of Sir2 did not significantly increase STRE-driven transcription activation,
we did not exclude the importance of Msn2/4 in elevated stress resistance of sir2 Δ
mutants. PKA and Tor controlled the transcription activity of Msn2/4 through regulating
its cellular localization. Msn2 and Msn4 are present in cytosol and translocate to the
nucleus under stress condition or caloric restriction to activate the transcription of stress
response genes. By using GFP tagged Msn2 expression plasmid, we tested if Sir2
affected the localization of Msn2 protein. We transformed wild type and sir2 Δ mutants
with this plasmid and visualized GFP signal under fluorescent microscope with log phase
and chronological aging day1 and day3 cells. Msn2 were mostly localized in nucleus in
exponential growing phase in both wild type and sir2 Δ mutants. On day3, the majority of
cells showed a cytoplasmic localization of Msn2 (Figure 2.7). Stronger GFP signals were
observed in sir2 Δ mutants, suggesting Sir2 mayregulate Msn2 expression instead of
localization. However, the data was not quantitative. Although microarray data analysis
did not reveal an increase in Msn2 expression at mRNA level in cells with Sir2
deficiency, expression of Msn2 and Msn4 at both transcriptional and translational level
during chronological aging are needed to be tested in order to elucidate the role of Sir2 in
regulating stress responses.
42

2.3.2 Sir2 deficiency promotes genomic stability through homologous
recombination related repair

Figure 2.8 Lack of Sir2 activity promoted genomic stability during yeast chronological
aging. (A) Mutation frequency over time in the CAN1 gene (measured as Can
r

mutants/10^6 cells, n >10). (B) Gross chromosomal rearrangement (GCR) frequency
(Can
r
5FOA
r
mutants/10^8 cells, n >=3). Age-dependent homologous (C, E) and
homeologous (D) recombination frequency measured as His + /10^6 cells. (n>=3)

By using the CAN1 gene mutation assay, Fabrizio et al found that mutation frequency of
the sir2 Δ mutant was higher than that of wild-type cells at early stationary phase (days 1–
3) but did not increase till day7. Deletion of Sir2 in the long-lived sch9 Δ mutant further
1 3 5 7 9 11 13 15 17 19 21 23
0
3
6
6
36
66
day
Mutation Frequency
(CAN
r
mutants/10^6 cells)
1 3 5 7 9 11
0
1
2
3
4
5
6
7
WT(DBY746)
sir2Δ
sch9Δ
sch9Δsir2Δ
day
GCRs Frequency
(Can
r
5FOA
r
mutants/10^8 cells)
1 3 5 7 9 11 13 15
0
20
40
60
60
560
1060
1560
Day
Inverted-repeat recombination
100% homologous substrate
(HIS
+
/ 10
6
cells)
1 3 5 7 9
0.00
0.25
0.50
0.75
1.00
WT (DBY746)
sir2Δ
sch9Δ
sch9Δsir2Δ
Day
Inverted-repeat recombination
91% homeologous substrate
(HIS
+
/ 10
6
cells)
1 3 5 7
0
250
500
750
1000 WT (DBY746)
sir2Δ
sir2Δ pSIR2
Day
Inverted-repeat recombination
100% homologous substrate
(HIS
+
/ 10
6
cells)
A B
C D
E
43

extended the low mutation frequency period. However, the mechanism through which
Sir2 promotes genomic instability during chronological aging is unknown.

First, we repeated the canavanine resistance mutation (Can
r
) assay, which detected
mutations that abolished Can1 function (Chen and Kolodner 1999). An age-dependent
spontaneous mutation frequency increase was observed in the wild-type (DBY746) cells
in agreement with the data shown by Fabrizio et al (Figure 2.8A). The sir2 Δ mutant did
not show a significant age-dependent increase through the whole life span but we did not
see an increase of mutation frequency of sir2 Δ mutants on day1 and day3 as shown by
Fabrizio et al. The deletion of SCH9 reduced and delayed the age-dependent increase of
spontaneous Can
r
mutation frequency (Fig. 2.8A; Fabrizio et al. 2005) and deletion of
Sir2 in sch9 Δ mutants remained constantly lower spontaneous mutation frequency till late
stationary phase day23, which supported the hypothesis that Sir2 promoted genomic
instability. Since Sir2 was involved in DNA double-strand breaks repair (Lee, Paques et
al. 1999), we looked at gross chromosomal rearrangements (GCRs) mutation, which
often are generated after double stranded breaks (Madia et al. 2008). Consistent with the
age-dependent increase in Can
r
mutations, wild type cells showed increased GCRs during
chronological aging. The deletion of SIR2 did not cause a significant increase in GCRs
which remained relatively constant during chronological aging (Figure 2.8B). Deletion of
Sir2 in sch9 Δ mutant did not further increase or decrease the GCRs frequency. It is worth
mentioning that only about 4% of the wild-type cells survived to day 11 (Fig. 2.1C),
which suggests that various spontaneous mutations including GCRs dramatically increase
44

the small subpopulation of old cells surviving to advanced ages. The inhibited increase in
CAN
r
mutation and GCRs in sir2 Δ mutant indicated the promotion of genomic instability
by Sir2.

Then we measured the frequency of homologous and homeologous recombinational
events in wild-type and sir2 Δ mutants. The error free homologous recombination may
help maintain genomic stability and the erroneous homoelogous recombination might
contribute to both Can
r
mutations and GCRs during aging. In fact, the erroneous
recombination has been shown to play a major role in GCR occurrence in sgs1 mutants
during growth phase (Myung, Datta et al. 2001). We integrated wild type (DBY746),
sir2 Δ, sch9 Δ and sch9 Δsir2 Δ mutants with an inverted-repeat substrate with 100 or 91%
homology, in which we could measure the frequency of recombination events (Datta,
Adjiri et al. 1996). There was a dramatic increase of homologous recombination
frequency in sir2 Δ mutants, which was about 30 times of that in wild type (Figure 2.8C).
However, no remarkable elevation of the erroneous homeologous recombination
frequency was detected in sir2Δ mutants compared to wild type (Figure 2.8D).
Overexpression of Sir2 with plasmid carrying functional Sir2 greatly reduced the
homologous recombination rate (Figure 2.8E), which supported the unusual high
homologous recombination frequency in sir2 Δ mutants and excluded the possibility that
this phenomena was due to other spontaneous mutations in the strain. During
chronological aging, the frequency of homologous recombination remained stable up to
day5 for wild type and slightly increased afterwards while sir2 Δ mutants displayed a
45

constantly high frequency. We also observed in wild type cells an age-dependent increase
of homeologous recombination frequency, which likely contributed to the increase of
Can
r
mutations and GCRs during chronological aging (Figure 2.8A, B)

Figure2.9 Deletion of Sir2 promotes genomic stability in the presence of oxidative stress
and DNA alkylation treatment. (A, B) Cells were treated with H
2
O
2
(2 or 5mM) on day 1
and day 3, mutation frequencies were measured from day1 to day7 (C, D) Cells were
treated with 0.005% MMS on day 1 (0 hour) and monitored on day3. N>=3. Bar was
shown as mean+SEM. Star denotes p<0.05.

Lack of Sch9 remarkably inhibited both homologous and homeologous recombination
frequency but no age-dependent increase in mutations was observed, in agreement with
the data shown by Madia et al that lack of Sch9 greatly reduced the homologous and
homeologous recombination frequency of sgs1 Δ mutant. Deletion of SIR2 in sch9 Δ
3 5 7
0
10
20
30
40
50
WT
sir2Δ
WT 2mM H2O2
sir2Δ 2mM H2O2
WT 5mM H2O2
sir2Δ 5mM H2O2
Day
Mutation Frequency
Can
r
mutants/10^6 cells
3 5 7 9
0
5
10
15
20
25
sch9Δ
sch9Δsir2Δ
sch9Δ 5mM H2O2
sch9Δsir2Δ 5mM H2O2
Day
Mutation Frequency
Can
r
mutants/10^6 cells
0 48
0
2
4
6
8
WT
sir2Δ
WT 0.005% MMS
sir2Δ 0.005% MMS
Hours
Mutation Frequency
Can
r
mutants/10^6 cells
0 48
0
1
2
3
4
5
sch9Δ
sch9Δsir2Δ
sch9Δ 0.005%MMS
sch9Δsir2Δ 0.005%MMS
Hours
Mutation Frequency
Can
r
mutants/10^6 cells
A B
C D
46

mutants increased the frequency of homologous recombination events 2-3 fold but did
not changed the homeologous recombination frequency, suggesting that the error free
homologous recombination might contribute to the further reduced Can
r
mutation
frequency in the sch9 Δsir2 Δ mutant.

To test the effects of genome protection by deletion of SIR2, we treated WT and sir2 Δ
mutants with H
2
O
2
(2mM and 5mM) and monitored the CAN
r
mutation frequency.
Hydrogen peroxide treatment greatly induced CAN1 gene mutation with 3-fold increase
under low concentration (2mM) and 11-fold increase under high concentration (5mM) of
H
2
O
2
in wild type (Figure 2.9A). In the severe oxidative stress environment, lack of Sir2
still caused a notably lower mutation frequency (marginally significant) after the
treatment (Figure 2.9A), indicating the protective effect by deletion of Sir2. We treated
sch9 Δ and sch9 Δsir2 Δ mutants with 5mM H
2
O
2
as well. The CAN1 gene mutation
frequencies in both mutants were much lower than those of wild type after treatment and
deletion of Sir2 in Sch9 deficient cells showed a trend for inhibiting genomic
mutagenesis but not statistically significant (Figure 2.9B). These results indicate that lack
of Sir2 promotes cellular protection against oxidative stress and probably protects the
genome directly, resulting in less damage in DNA and a lower mutation frequency.
Deletion of Sch9 had a strong protective effect against oxidative stress, which explains
the lack of a significant decrease in CAN
r
frequency in sch9 Δsir2 Δ mutants.

47

We also tested if Sir2 deficiency could protect cells against double strand breaks. Cells
were treated with the DNA alkylating agent Methyl methanesulfonate (MMS), which is
also a carcinogen. MMS methylates DNA on N
7
-deoxyguanine and N
3
-deoxyadenine,
which results in double-stranded DNA breaks and a stalled replication fork. Homologous
recombination-deficient cells show high sensitivity to MMS and have difficulty in
repairing the damaged replication forks after MMS treatment (Lundin, North et al. 2005).
There was a pronounced increase in CAN1 gene mutation after 48 hours upon MMS
treatment for both wild type and sir2 Δ mutants (Figure 2.9C). Instead of sensitizing cells
to MMS, the Can
r
mutation frequency in Sir2 deficient cells was only half of that of wild
type. Consistent with H
2
O
2
treatment, sch9 Δ and sch9 Δsir2 Δ mutants had similar level of
Can
r
mutation frequency, which was less than that in wild type. These results supported
the protective effect of Sir2 deficiency against different forms of DNA damage (Figure
2.9D).
48

Figure 2.10 Msn2, Msn4 and Gis1 do not mediate the enhanced genomic protection of
Sir2 deficient strain. Mutation frequency over time in the CAN1 gene (measured as Can
r

mutants/10^6 cells, n >=3). Strains are shown (A) Wild type, sir2 Δ, msn2/4 Δ,
sir2 Δmsn2/4 Δ; (B)Wild type, sir2 Δ, gis1 Δ, sir2 Δgis1 Δ; (C) Wild type, sir2 Δ,
msn2/4Δgis1Δ, msn2/4 Δgis1Δsir2 Δ;

The next question we asked is whether the enhanced protection of genomic stability in
Sir2 deficient cell was mediated by Msn2/4 and Gis1 as the protection against thermo and
oxidative stresses. Therefore, we measured the age-dependent spontaneous Can
r
mutation
frequency of msn2/4Δ, gis1 Δ mutants combined with Sir2 deficiency. Deletion of Msn2/4
or especially Gis1 showed the trend to increase the mutation frequency in wild type but
not in sir2 Δ mutants (Figure 2.10A, B). This suggested that the cellular protection against
genomic mutagenesis in Sir2 deficient strain was not dependent on the activity of Msn2/4
1 3 5 7 9
0
2
4
6
8
WT(DBY746)
sir2Δ
gis1Δ
gis1Δsir2Δ
day
CAN1 mutation freq.
/10^6 cells
1 3 5 7 9
0
2
4
6
8
WT(DBY746)
sir2Δ
msn2/4Δgis1Δ
msn2/4Δgis1Δsir2Δ
day
CAN1 mutation freq.
/10^6 cells
1 3 5 7 9
0
2
4
6
8
WT(DBY746)
sir2Δ
msn2/4Δ
msn2/4Δsir2Δ
day
CAN1 mutation freq.
/10^6 cells
A B
C
49

or Gis1. Interestingly, the triple msn2/4 Δgis1Δ and quadruple msn2/4Δgis1Δsir2 Δ mutant
exhibited a decreased and unprogressive mutation frequency during aging, implying other
regulatory pathways may be activated to maintain the genomic stability. This result
should be confirmed by other mutation assays (Figure 2.10C).

To examine if the enhanced homologous recombination in Sir2 deficient cell was
dependent on the homologous recombination machinery, we deleted Rad51 in wild type
and sir2 Δ mutants. Rad51 is a strand exchange protein, involved in the recombinational
repair of DNA double-strand breaks (DSBs). Rad51 is the homolog of Dmc1p and
bacterial RecA protein, which forms a helical filament with DNA that searches for
homology in recombinational repair (Shinohara, Ogawa et al. 1992; Sung 1994; Paques
and Haber 1999). Deletion of Rad51 did not reduce homologous recombination
frequency in wild type (Figure 2.11A). It is probably because double strand breaks can be
repaired by multiple pathways in yeast, whereas Rad51 is only responsible for synthesis-
dependent strand annealing (Paques and Haber 1999). However, deletion of Rad51 in
sir2 Δ mutants decreased the particularly high level of homologous recombination,
suggesting it partially mediated the increased homologous recombination caused by Sir2
deletion. The rad51 Δsir2 Δ double deletion mutant still had 3-4 fold of homologous
recombination rate of wild type (Figure 2.11A).
50

Figure 2.11 Homologous recombination machinery is required for the high homologous
recombination rate in cells lack of Sir2. (A) Age-dependent homologous recombination
frequency measured as His + /10^6 cells. (n>=4). (B)Chronological life span (CLS) of
wild type (DBY746), sir2 Δ, rad51 Δ and rad51 Δsir2 Δ (n>=4). (C)Mutation frequency
over time in the CAN1 gene (measured as Can
r
mutants/10^6 cells, n >=4).(D) The
mutation frequency of CAN1 gene during chronological aging.(n>=3).(E)
B)Chronological life span (CLS) of wild type(DBY746), sir2 Δ, rad54 Δ and
rad54 Δsir2 Δ(n>=3) (F) Base substitutions (trp1-289 reversion) frequency (measured as
Trp
+
revertants/10^8 cells, n>=3).

To understand the contribution of the excessive homologous recombination events to
genomic stability, we measured the CAN
r
mutation frequency of rad51 Δ and
rad51 Δsir2 Δ mutants. Deletion of Rad51 dramatically increased the CAN
r
mutation
1 3 5 7 9 11
0
50
100
150
700
1300
1900
WT
sir2Δ
rad51Δ
rad51Δsir2Δ
Day
Inverted-repeat recombination
100% homologous substrate
(HIS
+
/ 10
6
cells)
1 3 5 7 9 11
0.0
2.5
5.0
5
45
85
85
195
305
WT
sir2Δ
rad51Δ
rad51Δsir2Δ
Day
Mutation Frequency
(CAN
r
mutants/10^6 cells)
1 3 5 7 9 11 13 15 17 19
0
50
100
WT
sir2Δ
rad51Δ
rad51Δsir2Δ
Day
Survival (%)
1 3 5 7 9 11
0.0
2.5
5.0
5
45
85
85
195
305
WT
sir2Δ
rad54Δ
rad54Δsir2Δ
Day
Mutation Frequency
(CAN
r
mutants/10^6 cells)
1 3 5 7 9 11
0
50
100
100
1100
2100
3100
4100
WT
sir2Δ
rad54Δ
sir2Δrad54Δ
Day
Mutation Frequency
(Trp
+
revertants/10^8)
1 3 5 7 9 11 13 15
0
25
50
75
100
125
WT
sir2Δ
rad54Δ
rad54Δsir2Δ
Day
Survival (%)
A B
C D
E F
51

frequency which was about 12 times of wild type, suggesting that the Rad51 mediated-
double strand breaks repair was essential for maintaining genomic stability (Figure
2.11C). Interestingly, the rad51 Δsir2 Δ double deletion mutant had even higher mutation
frequency, about 2.5 times of that in rad51 Δ mutant and an age-dependent increase in the
mutation frequency was observed as well (Figure 2.11C). This result indicated that lack
of Sir2 promoted double strand breaks repair through homologous recombination. It is
worth to mention that lack of Rad51 did not reduce chronological life span although the
CAN
r
mutation frequency was particularly high which implied the uncoupling of life
span and genomic stability (Figure 2.11B). To confirm these observations, we mutated
another double-strand breaks recombinational repair gene Rad54, a DNA-dependent
ATPase, which stimulates strand exchange by modifying the topology of double-stranded
DNA (Clever, Interthal et al. 1997; Petukhova, Van Komen et al. 1999). Both Rad51 and
Rad54 belong to Rad52 epistasis group (Game and Mortimer 1974), involved in double
strand breaks repair. Rad54interacts with Rad51 and ssDNA to stimulate homolgous
pairing of DNA (Clever, Interthal et al. 1997). Consistent with what we observed for
rad51 Δ mutant, deletion of Rad54 caused remarkably elevation of CAN
r
mutation
frequency, 12-20 times of that in wild type (Figure 2.11D) and did not affect
chronological survival (Figure 2.11E). The rad54 Δsir2 Δ mutant had similar level of
mutation frequency as rad54 Δ single mutant. We preformed another mutation assay, in
which we measured point mutation frequency (trp1-289 reversions, trp
-
→ Trp+) (Madia,
Gattazzo et al. 2008). Lack of Sir2 significantly decreased the TRP reversion frequency
in agreement with the CAN1 mutation frequency. Cells without Rad54 activity greatly
52

increased the point mutation rate (Figure 2.11F) and deletion of Sir2 only slightly
reduced the particularly high mutation frequency of rad54 Δ mutants.
A

B

C

Figure 2.12 Thermal and Oxidative Stress Resistance and Methyl methanesulfonate
(MMS) treatment of cells deficient in Sir2, Rad51 and Sch9. (A) Chronological aging
day1 and day3 Wild type(DBY746), sir2 Δ, rad51 Δand sir2 Δrad51 Δmutants were
exposed to heat shock (55
o
C) or H
2
O
2
(100-150mM) for 30min. (B) The same strains
were treated with 0.1% MMS on day1 and 0.2% MMS on day3. (C) The sch9 Δ and
sch9 Δsir2 Δ mutants were treated with 0.2% MMS on day3.
53

Our data has shown that the double strand break recombinational repair machinery was
import for genomic stability. We also asked if it played a role in stress resistance as well.
Therefore, we treated rad51 Δ and sir2 Δrad51 Δ mutants with thermo and oxidative
stresses. Lack of Rad51 did not sensitize cells to heat shock but caused cell vulnerable to
oxidative stress (Figure 2.12A). This was probably because thermal stress increased the
accumulation of malfunctional proteins while oxidative stress also resulted in DNA
damage in addition to impair protein functions. Furthermore, the enhanced stress
resistance of Sir2 deficient cells was not affected by the deletion of Rad51 (Figure 2.12A),
indicating that the stress resistance and DNA repair are independent in sir2 Δ mutants.
These data support the hypothesis that lack of Sir2 renders the genome less vulnerable to
damage induced by various stresses. Because the RAD52 epistasis group is defective in
the repair of DNA damage caused by MMS (Symington 2002) and we have shown a
protection against MMS treatment by deletion of Sir2, we treated the sir2 Δrad51 Δ mutant
with MMS in order to test if the protection is Rad51 dependent. Consistent with what
shown by other group, cells deficient in Rad51 were hypersensitive to MMS (Figure
2.12B). The sir2 Δ mutant displayed a 10-fold resistance to MMS treatment compared to
wild type cells (Figure 2.12B). There was remained resistance to MMS in sir2 Δrad51 Δ
mutant under low concentration (1%) but not high concentration (2%), which suggested
the limited capacity of cells lack of Sir2 to repair the DSBs caused by MMS. In addition,
we examined the response of sch9 Δ mutants to MMS treatment. Interestingly, cells that
lack Sch9 display enhanced genomic stability (Fabrizio, Liou et al. 2003; Madia,
Gattazzo et al. 2008) by decreasing error prone repair (Madia, Wei et al. 2009) but the
54

sensitivity to MMS was observed in sch9 Δ mutants and deletion of SIR2 only slightly
improved the survival after MMS treatment (Figure 2.12C). This is probably because
homologous recombination was significantly inhibited by deletion of SCH9, which
limited the capacity to repair DSBs (Figure 2.8C).

In addition to homologous recombination, we also investigated other DNA repair
machineries. Previously, Madia et al reported that attenuation of age-dependent increases
in point mutation of cells lacking Sch9 is dependent on reduced REV1 expression. Rev1
forms a complex with the subunits of DNA polymerase zeta, Rev3 and Rev7, involved in
repair of abasic sites and adducted guanine by translesion synthesis during post-
replication repair and double-strand break repair (Nelson, Lawrence et al. 1996; Acharya,
Johnson et al. 2006; Hirano and Sugimoto 2006). Since Rad51 only partially mediated
the elevation of recombinational repair of DSBs, we tested whether the error prone repair
also played a role in the Sir2 promoted genomic instability. As shown by Madia et al,
deletion of Rev1 significantly decreased the CAN
r
mutation frequency but reduced the
chronological life span, which indicated the error prone repair was important for cell
survival. Interestingly, Sir2 deficiency in rev1 Δ mutants rescued the shortened life span
but remained low CAN
r
mutation frequency (Figure 2.13A, B), indicating that lack of
Sir2 promoted a error-free repair mechanism to compensate the impaired polymerase zeta
dependent error-prone DNA repair. Deletion of Rev1 further decreased the CAN
r

mutation frequency in sch9 Δsir2 Δ mutant by half (Figure 2.13B), suggesting that the Sir2
promoted genomic instability was independent of Rev1. Consistent with the results
55

shown by Madia et al, lack of Rev1 increased GCRs events but not in sir2 Δ mutants
(Figure 2.13C).

E

Figure 2.13 Deletion of Sir2 restored chronological lifespan of rev1 Δ mutants but did not
increase mutation frequency. (A) Chronological life span (CLS) of Rev1 deletion mutants.
(B) Mutation frequency over time in the CAN1 gene (measured as Can
r
mutants/10^6
cells, n >=3) (C) Gross chromosomal rearrangement (GCR) frequency (Can
r
5FOA
r

mutants/10^8 cells, n>=3). (D) Age-dependent homologous recombination frequency
measured as His + /10^6 cells. (n>=3) (E) Stress resistance assays with Rev1 deletion
mutants.
0 2 4 6 8 10 12 14 16
0
50
100
150
200
WT (DBY746)
sir2Δ
rev1Δ
sir2Δrev1Δ
sch9Δsir2Δ
sch9Δsir2Δrev1Δ
day
Survival (%)
1 3 5 7 9 11 13 15
0
3
6
6
36
66
WT (DBY746)
sir2Δ
sir2Δrev1Δ
sch9Δsir2Δ
sch9Δsir2Δrev1Δ
day
Mutation Frequency
(CAN
r
mutants/10^6 cells)
A B
1 3 5 7 9 11
0
1
2
3
4
5
6
7
WT(DBY746)
sir2Δ
rev1Δ
sir2Δrev1Δ
day
GCRs Frequency
(Can
r
5FOA
r
mutants/10^8 cells)
1 3 5 7
0
50
100
150
150
650
1150
1650
WT (DBY746)
sir2Δ
rev1Δ
sir2Δrev1Δ
Day
Inverted-repeat recombination
100% homologous substrate
(HIS
+
/ 10
6
cells)
C D
56

We also examined the effect of Rev1 on homologous recombination. A remarkably
elevation in homologous recombination frequency was seen in rev1 Δ mutant, which was
about 2-3 fold of that in wild type and a further increase of the particular high
homologous recombination frequency was observed the sir2 Δrev1 double deletion mutant
(Figure 2.13D). This elucidated that deletion of Rev1 induced a compensatory
recombinational repair mechanism, which was Sir2 independent. The stress resistance
assay revealed that Rev1 played minor role in the stress response (Figure 2.13E) which
justified the indirect correlation between genomic stability and stress response. To
demonstrate if the compensatory repair was homologous recombination machinery
dependent, we tried to generate rev1 Δrad51 Δ and sir2 Δrev1 Δrad51 mutants. But due to
the importance of these DNA repair genes, we did not successfully get the desired
mutants, which supported the compensatory effects between the two DNA repair
mechanisms.

2.3.3 Sir2 and Ras signaling
Previously, we have collaborated with computational biologists to analyze the gene
expression files of ras2 Δ, sch9 Δ and sch9 Δsir2 Δ mutants and compared then to those of
wild type cells. Each strain had triplicates and was collected on day 2.5 to get enough
mRNA for microarray experiment, as many cells became hypometabolic following a
dramatic decrease in transcriptional activity at older age. For one part of the analysis, we
made inference about the modifications of biological activities using gene subsets defined
by Gene Ontology (GO), based on the expression fold changes (Cheng, Fabrizio et al.
57

2007). The p value denoted the significance of the enrichment of the up- or down
regulated genes in each group and the q value represents the false discovery rate (Cheng,
Fabrizio et al. 2007). Among the significantly up- and down-regulated GO group, we
found that the pattern of gene expression was very similar between ras2 Δ and sch9 Δsir2 Δ
mutants (Table1). There were several overlaps in modification of activity of gene subsets
between sch9 Δ and ras2 Δ mutants as well but not as pronounced as those in ras2 Δ and
sch9 Δsir2 Δ mutants. Therefore, we proposed that there was genetic connection between
Ras2 and Sir2. Considered that the double deletion of Sch9 and Ras2 resulted in 10 times
life span extension when combined with caloric restriction, we hypothesized that Sir2
modified Ras2 activity, resulting in further life span extension in CR and cells lack of
nutrients sensing signaling.

58

Table 2.1. Up and Down-regulation of TIGO categories relating to transcription.

Positively Regulated GO Group
sch9 ∆/wt3 ras ∆/wt3 sch9sir2 ∆/wt3
p-value q-value p-value q-value p-value q-value
cytosolic large ribosomal subunit 0 0 2E-12 2E-10 2E-12 2E-10
cytosolic small ribosomal subunit 0 0 7E-09 6E-07 1E-09 1E-07
monosaccharide catabolism 0.0013217 0.0200885 9E-06 4E-04 2E-04 4E-03
Negatively Regulated GO Group
mitochondrial large ribosomal subunit 2E-19 3E-17 1E-20 4E-18 8E-17 1E-14
mitochondrial small ribosomal subunit 7E-13 5E-11 5E-14 4E-12 2E-12 1E-10
DNA-directed RNA polymerase II\,
holoenzyme 2E-05 2E-04 5E-10 2E-08 2E-10 1E-08
proteasome complex 4E-04 3E-03 1E-08 5E-07 5E-06 9E-05
major (U2-dependent) spliceosome 2E-02 5E-02 7E-05 7E-04 1E-04 9E-04
histone acetyltransferase complex 2E-02 5E-02 2E-05 2E-04 8E-05 8E-04
mitochondrial inner membrane 3E-16 3E-14 3E-09 1E-07 6E-08 2E-06
general RNA polymerase II transcription factor
activity 2E-02 5E-02 1E-06 3E-05 5E-05 5E-04
protein transporter activity 3E-04 2E-03 1E-02 4E-02 5E-03 2E-02
transcription initiation from RNA polymerase II
promoter 3E-02 7E-02 9E-06 1E-04 5E-04 3E-03
mRNA-nucleus export 5E-02 9E-02 1E-04 1E-03 3E-04 2E-03
histone modification 2E-03 8E-03 7E-06 1E-04 4E-05 5E-04
35S primary transcript processing 2E-03 9E-03 4E-03 2E-02 5E-05 6E-04
transcription from RNA polymerase III
promoter 2E-02 5E-02 6E-03 2E-02 2E-03 8E-03
biopolymer methylation 1E-03 6E-03 1E-02 4E-02 1E-04 1E-03
processing of 20S pre-rRNA 3E-04 2E-03 4E-02 8E-02 1E-04 1E-03
mitochondrion organization and biogenesis 7E-05 7E-04 5E-06 8E-05 4E-05 5E-04
mRNA catabolism 7E-02 1E-01 1E-03 5E-03 2E-02 5E-02
nucleotide-excision repair 1E-01 2E-01 2E-03 8E-03 2E-02 5E-02
positive regulation of transcription from RNA
polymerase II promoter 1E-01 2E-01 4E-05 5E-04 5E-03 2E-02
rRNA modification 5E-02 9E-02 2E-01 3E-01 2E-02 5E-02
protein-nucleus export 4E-02 8E-02 7E-03 2E-02 2E-02 5E-02
translational initiation 2E-02 5E-02 4E-02 8E-02 2E-02 5E-02
protein-mitochondrial targeting 8E-06 1E-04 4E-04 3E-03 9E-04 5E-03
aerobic respiration 3E-08 9E-07 1E-06 3E-05 1E-04 1E-03
oxidative phosphorylation 7E-07 2E-05 2E-04 1E-03 8E-04 5E-03
electron transport 1E-04 1E-03 4E-03 2E-02 1E-01 2E-01
59

D

Figure 2.14 Deletion of Sir2 further increased the life span of long-lived ras2 Δ mutants.
(A) CLS of wild type, sir2 Δ, ras2 Δ, ras2 Δsir2 Δ. (B) Mutation frequency over time in the
CAN1 gene (measured as Can
r
mutants/10^6 cells, n >=6). Age-dependent homologous
(C) recombination frequency measured as His + /10^6 cells (n>=3). (D) WT, sir2 Δ,
ras2 Δ and ras2 Δsir2 Δ were exposed to heat stress (55
o
C) or to H
2
O
2
(500 mM) for 30min.

To test this hypothesis, we first generated the ras2 Δsir2 Δ double deletion mutants.
Similarly to sch9 Δ mutants, deletion of Sir2 in Ras2 deficient cell further extended
chronological life span, in agreement with the data shown by Fabrizio et al that lack of
Sir2 prolonged life span of the mutant with deficiencies in Cyr1: a Ras2 effector (Figure
0 2 4 6 8 1012141618202224262830323436
0
25
50
75
100
125
WT(DBY746)
sir2Δ
ras2Δ
ras2Δsir2Δ
Day
Survival (%)
1 3 5 7 9 11 13 15
0
3
6
6
36
66
WT(DBY746)
sir2Δ
ras2Δ
ras2Δsir2Δ
Day
Mutation Frequency
(CAN
r
mutants/10^6 cells)
1 3 5 7 9 11
0
20
40
60
80
260
460
660
860 WT (DBY746)
sir2Δ
ras2Δ
ras2Δsir2Δ
Day
Inverted-repeat recombination
100% homologous substrate
(HIS
+
/ 10
6
cells)
A B
C
60

2.14A). This result did not favor our hypothesis since the life span of ras2 Δ mutants was
still modified by Sir2 deletion. We also looked at how Ras2 affected genomic stability by
measuring the CAN1 gene mutation frequency. Unlike the Sch9 deficiency, lack of Ras2
did not promote the major decrease in DNA mutagenesis as measured by CAN
r
mutation
frequency, suggesting a differential regulation of longevity by the Tor/Sch9 and
Ras/cAMP/PKA pathways. However, deletion of Sir2 remarkably lowered the mutation
frequency of ras2 Δ mutants, which might partially explain the prolonged life span of
ras2 Δsir2 Δ mutant (Figure 2.14B). To test the effect of Ras2 on homologous
recombination events, we measured the homologous recombination frequency as
described previously. Lack of Ras2 increased the frequency of homologous
recombination compared to that in wild type but decreased that in sir2 Δ mutants (Figure
2.14C). Furthermore, deletion of both RAS2 and SIR2 caused a hyperresistance to
extreme thermo and oxidative challenges (Figure 2.14D).

F
re
w
m
A
pr
an
igure 2.15
educed by d
wild-type (D
monoclonal a
A representat
rotein is sho
nd sir2 Δ mu
The protein
deletion of S
DBY746) an
anti-Ras anti
tive experim
own as well.
utant cells. Q
Ras2 Protein
Ras2 Protein
Normalized with GAPDH
n level of R
Sir2 in early
nd sir2 Δ m
body. The h
ment was sho
(B) Wester
Quantitative a
0.3
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Ras2 Protein
Normalized with GAPDH
0.3 1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Ras2 increa
y stationary p
mutant cells
house keepin
own and corr
rn blot analy
analysis of R
1.0 3.0 5.0
Day
.0 3.0 5
Day
sed with ch
phase. (A) W
s. Protein l
ng gene GAP
responding q
ysis of Ras2
Ras2 protein
0 7.0
WT(
sir2Δ
5.0 7.0

hronological
Western blo
levels were
PDH level se
quantitative
protein in w
was shown
(DBY746)
Δ
WT(W303)
sir2Δ
l aging and
t of lysates
e detected b
erved as a co
analysis of
wild-type (W
below.
61

d was
from
by a
ontrol.
Ras2
W303)
62

Interestingly, when we examined the Ras2 protein level, we found an accumulation of
Ras2 protein with age, implying a Ras2 signaling dependent cell death mechanism.
Although we did not see a genetic interaction between Ras2 and Sir2, we observed a
reduction of Ras2 protein level in cells lack of Sir2 in early stationary stage day1 and
day3 (Figure 2.15A). On day5 and day7, the amount of Ras2 protein detected by western
blot was similar between wild type and sir2 Δ mutants (Figure 2.15A). To confirm this
discovery, we measured the Ras2 protein during chronological aging in another yeast
background W303. Similar result was obtained in W303 strains in which the Ras2 protein
level was lower in Sir2 deficient cells compared to wild type, suggesting a potential role
of Sir2 in regulating Ras2 expression (Figure 2.15B).

Figure 2.16 Transcription level of Ras2. Quantitative measurements of Ras2 mRNA were
performed in wild type (DBY746) and sir2 Δ mutants using quantitative real time PCR.
Numbers are normalized with Actin mRNA level and adjusted by day1 wild type mRNA.

To examine how Sir2 regulates Ras2 expression, we checked the mRNA level of Ras2.
There was a significant decrease in Ras2 transcription in sir2 Δ mutants on day3 but not at
other stages, indicating the regulation by Sir2 was age dependent (Figure 2.16). Then we
0.3 1.0 3.0 5.0 7.0
0.0
0.5
1.0
1.5
WT
sir2Δ
Day
RAS2 mRNA
Normalized with Actin mRNA
Adjusted to Day1 WT
63

asked if Sir2 modified Ras2 protein at the post-translational level, since Sir2 had been
shown to directly deacetylate several stress-responsive factors, such as forkhead
transcription factor (FOXO1) and the p53 tumor suppressor. We proposed that
acetylation might promote stability of Ras2 protein. Immunoprecipitation was employed
to detect any posttranslational acetylation of Ras2 or any protein-protein interaction
between Ras2 and Sir2. Wild type and sir2 Δ mutant cells were collected on day3 and
immunoprecipitated with Anti-Ras antibody. This anti-body was very specific as no
signal was detected in Ras2 deletion mutant. Then western blot was performed with the
pull-down proteins. Anit-Ras antibody was utilized to confirm the enrichment of Ras2
protein and Anti-Sir2 was used to detect possible protein interaction (Figure 2.17). The
posttranslational modification of acetylation was detected by anti-acetylated lysine
antibody. We saw an enrichment of Ras2 protein after immunoprecipitation but we did
not find any acetylation or protein interaction signal, which disfavored the hypothesis
about the posttranslational modification of Ras2 by Sir2.

F
W
im
A

C
fr
W
W
th
ch
ex
igure 2.17 Im
Wild-type, si
mmunopreci
Anti-Sir2 or a
Caloric restri
rom yeast to
We grew wild
Western blot
hat CR red
hronological
xtension (Fig
mmunoprec
ir2 Δ and ra
ipited with a
anti-acetylat
ction (CR) h
mouse. We
d type cells i
was perform
duced the
l aging, su
gure 2.18).
ipitation doe
as2 Δmutant
anti-Ras antib
ed lysine an
has been sho
asked if CR
in 0.5% SDC
med to exam
generation
uggesting a
es not reveal
cells were
body and we
tibody.
own to incre
R could atten
C medium an
mine the pro
and inhibi
potential m
l posttransla
collected o
estern blot w
ease life spa
nuate the age
nd collected
otein level o
ited the ac
mechanism
ational deace
on day3. C
were perform
an of wide ra
e-dependent
d cell sample
of Ras2. The
ccumulation
for CR in
etylation of R
Cell lyastes
med with ant
ange of orga
increase of R
s every othe
e results rev
of Ras2
nduced life
64

Ras2.
were
ti-Ras,
anism
Ras2.
er day.
vealed
with
span
65

Figure 2.18 Quantification of Ras protein level under caloric restriction. Wild type
(DBY746) cells grew in 2% or 0.5% SDC medium and collected on d1 to d9 in two days
interval. Western blot were performed and Ras protein levels were adjusted to GAPDH.

In summary, Sir2 modulated Ras2 protein level, although direct protein interaction or
posttranslational acetylation evidence lacked. Caloric restriction was able to down-
regulate Ras signaling by inhibiting Ras2 expression, raising the possibility that CR, Sir2
and Ras protein stability and levels may be connected.

2.4 Discussion
Sirtuins, a family of deacetylases, have been intensively studied for decades because of
its crucial role in numerous cellular processes. Sir2 is the founding member of the sirtuin
family (Landry, Sutton et al. 2000; Smith, Brachmann et al. 2000) and its role in aging
d1 d3 d5 d7 d9 d1 d3 d5 d7 d9
2%SDC 0.5%SDC
Anti-RAS
Anti-GAPDH
1 3 5 7 9
0.0
0.1
0.2
0.3
0.4
2% SDC
0.5% SDC
Day
Ras2 Protein
Normalized with GAPDH
66

was first demonstrated in a yeast replicative aging study (Kaeberlein, McVey et al. 1999;
Imai, Armstrong et al. 2000; Lin, Defossez et al. 2000). Lack of Sir2 reduced replicative
life span while overexpression of Sir2 is sufficient to increase replicative life span
(Kaeberlein, McVey et al. 1999). It was also suggested that Sir2 is required for CR-
mediated replicative life span extension (Lin, Defossez et al. 2000).The mechanism of
Sir2-promoted replicative longevity may be through repression of homologous
recombination at ribosomal DNA (rDNA), which induces formation of
extrachromosomal rDNA circles (ERCs). ERCs accumulation is considered to be the
primary mechanism of aging, although this mechanism has been challenged recently
(Lindstrom, Leverich et al. 2011). In contrast to the anti-aging role of Sir2, our lab
reported the opposite role for Sir2 in chronological aging, where Sir2 blocks the extreme
longevity caused by mutations in the nutrient sensing Tor/Sch9 and Ras/cAMP/PKA
signaling pathways and caloric restriction (Fabrizio, Gattazzo et al. 2005). The
discrepancy between the roles of sir2 in yeast replicative and chronological life spans is
probably due to the different systems of identifying aging phenotype. Cells in the
replicative aging model stay in an active growth phase, where most stress response genes
are repressed, while chronological aging measures the survival of cells in stationary
phase in which DNA damage and malfunctioning proteins accumulate. The Sir2-
regulated stress response genes are especially important for cell chronological survival. In
addition, rDNA instability and ECR accumulation are probably not a primary cause of
chronological aging.

67

Sir2 promotes silent chromatin formation at the mate type loci, HML and HMR, which in
turn repress HM-associated gene expression (Rine, Strathern et al. 1979). The
disassociation of Sir2 from HM loci is believed to be beneficial since the transient HM-
associated genes promote DNA repair (Lee, Paques et al. 1999). However, to determine
whether the Sir2 deletion phenotype is HM dependent, we generated the HMRa locus
deletion mutants. No difference in survival and stress resistance was observed, which
supported a pro-aging effect for Sir2. The enhanced stress resistance is one of the most
prominent phenotypes of sir2 Δ mutant. Although microarray data revealed that Sir2
regulated many stress response genes, it is unclear if regulation of stress response is
required for life span extension and by what means Sir2 impacts the expression of these
genes. Therefore we investigated if the upstream stress response transcription factors
mediate the increased stress resistance from lack of Sir2. Our lab has previously shown
that Msn2/4, Gis1 and the upstream kinase Rim15 are required for life span extension by
mutations in the TOR/SCH9 and RAS/cAMP/PKA signaling pathway as well as caloric
restriction, which is in agreement with the crucial role of Msn2/4 in replicative aging
under CR (Fabrizio, Pozza et al. 2001; Fabrizio, Liou et al. 2003; Medvedik, Lamming et
al. 2007; Wei, Fabrizio et al. 2008).

Our results suggest that Msn2/4 and Gis1 are required for the resistance to oxidative
stress associated with Sir2 deficiency. Removal of both Msn2/4 and Gis1 or Rim15
abrogates the advanced cellular protection against oxidative stress (Figure 2.2B). Cells
without functional Sir2, Msn2/4 and Gis1 still retain resistance to thermo stress, which
68

suggest that these transcriptional factors only partially mediate Sir2's effects. In addition,
Msn2/4 and Gis1 are not required for the normal life span of wild type cells but are
required for the extended survival caused by mutations in SCH9 and RAS2 and by CR,
which suggests that the increased stress response is not required for regular life span but
for extreme longevity. The further life span extension caused by Sir2 deficiency in the
long-lived mutants and CR was also attenuated by deletion of Msn2/4, Gis1 and Rim15
(Figure 2.4 C, D and E).

Gis1 regulates gene expression after glucose depletion after the diauxic shift phase, when
metabolism of yeast cells shifts from glucose fermentation to respiration with ethanol
oxidation (DeRisi, Iyer et al. 1997). Previously, Zhang et al also reported that a group of
genes significantly up-regulated upon starvation were Gis1 dependent and the
transcriptional reprogramming of carbon metabolism during the switch from growth to
stationary phase also required Gis1p (Zhang, Wu et al. 2009). Recent studies addressed
that activity and specificity of Gis1-dependent effects on the growth phase. The activity
of Gis1 is repressed during the exponential growth phase, whereas after the diauxic shift,
Gis1 targets genes with STRE or PDS motifs in the promoter region and predominantly
activates genes with PDS motifs (Orzechowski Westholm, Tronnersjo et al. 2012),
suggesting a cross talk between Msn2/4 and Gis1 in the regulation of gene expression. To
dissect the regulation of Msn2/4 and Gis1 by Sir2, we used a LacZ reporter gene with
either STRE or PDS elements in the promoter region. To our surprise, we did not see
significant increase in STRE-driven gene expression. Instead, an increase in PDS-driven
69

transcription activation was observed. This suggests that Sir2 represses Gis1's activity.
Notably, we observed that Gis1 deletion promoted STRE-driven transcription activation,
which indicated the repression of genes containing STRE elements by Gis1 and was
consistent with a recent finding that Gis1 suppress the expression of genes with STRE
motifs in the promoter regions (Orzechowski Westholm, Tronnersjo et al. 2012).Gis1 is
essential for the life span extension by Sch9 deficiency as shown by Min et al (Wei,
Fabrizio et al. 2008). Here we demonstrate that Gis1 is also crucial for the further life
span extension by deletion of Sir2 in a sch9 Δ mutant, since the sch9 Δsir2 Δ gis1Δ triple
mutants were not different from sch9 Δsir2 Δ double deletion mutants (Figure 2.4C).

Unexpectedly, when we overexpressed Gis1 to see if it promoted longevity, we found
that excessive Gis1 had toxic effects on cell survival (Figure 2.5). Zhang et al also
reported that overexpression of full length Gis1 in cells caused growth inhibition,
although the complete Gis1 protein had the largest potential in inducing the transcription
of stress response genes such as GRE1 (Zhang and Oliver 2010). Interestingly,
overexpression of the downstream Sod2 does not change the lifespan or age-related
pathology (Jang, Perez et al. 2009). As Gis1 induces and represses the expression of a
large set of genes, the level of Gis1 protein is probably under strict regulation. The
truncated form of Gis1 is less toxic (Zhang and Oliver 2010), indicating a domain
oriented mechanism. Detailed dissection of transcriptional repression and induction by
Gis1 is needed to understand the role of zinc finger transcription factors in cell survival
and longevity.
70

The Sir2 deficient cells are more tolerant to Gis1 overexpression (Figure 2.5), supporting
a role of Sir2 as a regulator of Gis1. In addition, a partial length of Gis1 was detected in
sir2 Δ mutants, which might explain the tolerance to excessive Gis1 toxicity. The multi
truncated form of Gis1 observed in our protein study may be caused by proteasome
mediated proteolysis (Zhang and Oliver 2010). Thus, we proposed that Sir2 might
suppress the degradation of Gis1 by proteasome by posttranslational modification.
However, no direct protein interactions or deacetylationshave been detected in our study,
indicating an indirect regulation of Gis1 protein stability by Sir2. It is worth mentioning
that rapamycin treatment also promotes the proteasome-mediated proteolysis (Zhang and
Oliver 2010), suggesting that TOR signaling also regulates Gis1's activity.

Although we did not see an increase of STRE-driven transcription activity in Sir2
deficient strain, results from the stress resistance assay did not exclude the possible link
between Sir2 and Msn2/4. The localization of Msn2/4 is important for its transcriptional
activity. Evidences suggest that TOR signaling promotes the cytoplasm retention of
Msn2/4 while CR promotes their nucleus relocalization (Beck and Hall 1999; Medvedik,
Lamming et al. 2007). In our MSN2-GFP experiment, we observed a nucleus localization
of MSN2 in the growth phase, which relocated to the cytoplasm during stationary phase
(Figure 2.7). A relatively strong signal without significant nucleus enrichment was seen
in sir2 Δ mutant at day3, indicating that Sir2 may not affect Msn2p localization. Instead, it
may regulate the expression or degradation of Msn2. Study of Msn2 at protein level may
be needed in future research.
71

The interaction between Sir2 and Ras2 was initially illuminated by microarray analyses.
Mutations in Ras/cAMP/PKA extend chronological aging (Fabrizio, Gattazzo et al. 2005;
Wei, Fabrizio et al. 2008). Recently, the RasGRF1 (Ras-guanine nucleotide exchange
factor) null mice were reported to have extended life span and delayed aging (Borras,
Monleon et al. 2011). Deletion of Sir2 further increases the chronological life span of
ras2 Δ mutant (Figure 2.14A). Notably, our data suggest increased Ras2p with age, which
is probably due to the reduced turnover of Ras2 protein with age. Furthermore, we found
that caloric restriction repressed the accumulation of Ras2p, suggesting that Ras2 down-
regulation may serve as a mediator of CR. We also found that Sir2 promoted Ras2
accumulation in early stationary phase but not in late stages. However, no
posttranslational modification of acetylation has been detected in Ras2p, suggesting an
indirect means of regulation of Ras2p stability by Sir2.

Sir2 was previously reported to maintain genomic stability by the repair of DNA double-
strand breaks through non-homologous end joining (NHEJ) (Lee et al. 1999). However,
the importance of the NHEJ function of Sir2 in aging was challenged (Kaeberlein et al.
1999). It has been shown that the Sir2 homolog Sirt6 suppresses genomic instability in
mice by promoting DNA repair (Mostoslavsky, Chua et al. 2006). Our data suggest that
lack of Sir2 maintains a constant rate of DNA mutations in contrast to the age-dependent
increase of mutagenesis in wild type cells (Figure 2.8A). In disagreement with the double
strand breaks repair by NHEJ, lack of Sir2 did not elevate the frequency of gross
chromosomal rearrangements predominantly caused by DSBs. Lack of Sir2 also
72

promoted genomic stability against oxidative stress and DNA alkylation induced
mutations (Figure 2.9A, C). There are additive effects on genomic instability by deletion
of both SCH9and SIR2. We speculate that this effect of Sir2 is dependent on homologous
recombination mediated repair, since we observed a particularly high homologous
recombination rate in sir2 Δ mutant which was in agreement with the role of Sir2 in
repressing recombination at rDNA (Kaeberlein, McVey et al. 1999). It is also proposed
that translocation of Sir2 proteins assist the DSBs repair by modifying the chromatin
structures (Lee, Paques et al. 1999). In contrast to the greatly increased homologous
recombination events, Sir2 deficiency does not promote error prone homeologous
recombination, suggesting that deletion of Sir2 promotes error free repair. A recent study
reported that Sirt1 translocated to DSBs and was required for Rad51 recruitment to
complete DNA repair (Oberdoerffer, Michan et al. 2008). We found that in the absence
of Sir2, Rad51 was still able to perform homologous recombination (HR) and was
required for the enhanced HR events (Figure 2.11A). This discrepancy is probably caused
by redundant yeast sirtuins, which also function in homologous recombination.
Furthermore, Rad51-mediated recombinational repair is crucial for genomic stability
(Figure 2.11C), which supports the hypothesis that Sir2 promotes genomic stability
partially through error free recombinational repair. In addition, lack of Sir2 compensates
for the deficiency caused by the diminished error prone translesion repair in the rev1 Δ
mutant, suggesting the positive role that Sir2 plays in facilitating other forms of repair,
probably the recombinational repair.

73

In summary, our study explored the mechanisms through which Sir2 affects
chronological aging. We uncovered the genetic regulation of Msn2/4 and Gis1 by Sir2,
although direct evidence still lacked. We also pointed out the essential role of
recombinational repair for maintaining genomic stability and that the lack of Sir2 may
facilitate the recruitment of DNA repair machinery and their access to the damaged loci.
In addition, we found that Sir2 and caloric restriction regulate Ras protein levels. Our
study provides new insights for studying sirtuins in higher eukaryotes.

74

Chapter 3 Role of Sirt1 in Caloric Restriction
3.1 Introduction
Silent information regulator 2 (sir2) is a yeast nicotinamide adenine dinucleotide (NAD)-
dependent histone deacetylase, which deacetylates histone H3 and H4 in the presence of
NAD(Imai, Armstrong et al. 2000; Landry, Sutton et al. 2000). The Sir2 family
proteinsSirtuins, are conserved from bacteria to humans (Brachmann, Sherman et al.
1995; Frye 2000). Seven mammalian Sir2 homologs, named SIRT1-SIRT7, have been
identified, which are considered to be functionally related to Sir2 (Frye 2000). Among
the seven mammalian Sir2 homologs, SIRT1 shares the most amount of homology with
Sir2 (Frye 2000) and has been characterized more extensively. Similar to the deacetylase
function of Sir2, SIRT1 has been shown to modify chromatin and be involved in gene
silencing (Vaquero, Scher et al. 2004). In addition to the histone deacetylase function,
SIRT1 also deacetylaces or binds to a large number of nuclear and cytoplasmic proteins,
including the stress response forkhead transcription factors(FOXOs) (Daitoku, Hatta et al.
2004; van der Horst, Tertoolen et al. 2004), tumor suppressor p53 (Luo, Nikolaev et al.
2001; Vaziri, Dessain et al. 2001; Langley, Pearson et al. 2002) and NF- κB (Yeung,
Hoberg et al. 2004).

Calorie restriction (CR), a dietary regimen, has been well known to affect the lifespan or
age-dependent function in organisms ranging from yeast to mammals. Sir2 was suggested
to be required for CR to extend lifespan in yeast, C.elegans and flies (Lin, Defossez et al.
75

2000; Rogina and Helfand 2004; Wood, Rogina et al. 2004). However, the role of Sir2 in
CR-mediated lifespan extension has been the subject of heated debate (Kaeberlein,
Kirkland et al. 2004; Fabrizio, Gattazzo et al. 2005; Lee, Wilson et al. 2006). Despite
these controversies in lower organisms, SIRT1 has been shown to be involved in
regulating multiple essential cellular processes including glucose metabolism, stress
response and apoptosis (Luo, Nikolaev et al. 2001; Bordone, Motta et al. 2006).
Furthermore, SIRT1 has been reported to improve health aging and ameliorate the onset
of age-associated cancers (Herranz, Munoz-Martin et al. 2010). However, the role of
SIRT1 in CR-mediated lifespan extension in mammals remains unknown. Previously, we
have shown that CR does not increase life span of SIRT1 null mice. However, due to the
defects and early mortality displayed by SIRT1 null mice, it is not clear if SIRT1 is
important for the effect of CR on lifespan or is simply required for the metabolic mode
entered during CR. Here we studied the effect of SIRT1 in CR-mediated longevity using
a SIRT1 heterozygote knockout mouse model, which maintain relatively normal levels of
SIRT1 without masking the CR effect.

3.2 Materials and methods
Animals SIRT1
+/+
,
+/-
and
-/-
genotypes have been described previously (Li, Xu et al.
2008) . Three to five-month-old mice were single caged and fed ad libitum (AL) or
restricted to 60% of AL group (CR) with NIH-31 standard chow or the NIH31/NIA-
fortified food (7109, Harlan Teklad), respectively. Water was available ad libitum for all
mice. Criteria for euthanasia: when mice are observed to have lesions, clinical symptoms
76

or continued body weight loss, we informed university LAR (Laboratory Animal
Sources). LAR veterinarian will make determinations about mouse conditions and need
to perform euthanasia. The animal protocol has been approved by USC IACUC
(Institutional Animal Care and Use Committee).

Y-maze. Mice were tested for working memory using a Y-maze (arms 21 cm x 4 cm,
length x width with 40-cm walls). The test started placing the rodent in one of the arms of
the maze. The mouse was allowed to explore freely the environment for 8 minutes and
the total numbers of arm entries and arm choices were recorded. An arm choice was
defined as both fore- and hind-paws fully entering the arm. Spontaneous alternation
behavior (SAB) score was calculated as the proportion of alternations (an arm choice
differing from the previous two choices) to the total number of alternation opportunities
(Rosario, 2006, PMID: 17182789).

Elevated Plus Maze (EPM). Mice were tested for anxiety using an Elevated Plus Maze
(EPM). The EPM has the shape of a cross formed by two alternate open and two alternate
closed arms extending from a central platform, each arm measuring 30 cm in length and
15 cm in height. The test is based on rodent exploratory behavior, balanced by natural
rodent aversion against open space. The avoidance of elevated open arms is an indication
of the intensity of anxiety. During the test the mouse was placed onto the center field and
was allowed to freely explore the maze for 5 minutes, and the time spent in the open arms
77

was measured. More time spent in open arms corresponded to lower anxiety levels
(Jawhar, 2010, PMID: 20619937).

3.3 Results
It has been reported previously that mice lacking both copies of SIRT1 have a shorter
median lifespan and maximum life span compared to wild type mice on an ad libitum diet
or diet with a 40% reduction in calories (Boily, Seifert et al. 2008; Li, Xu et al. 2008).
However, the role of SIRT1 in the effects of CR is not clear since SIRT1 is essential for
normal embryogenesis and development. In an inbred background, SIRT1null mice are
smaller than normal and died shortly after birth during the early postnatal period. In an
outbred background, the SIRT1 null mice grow normally but are sterile and have
craniofacial abnormalities and eyelid inflammation (McBurney, Yang et al. 2003). These
deficiencies observed in SIRT1 null mice might outweigh the beneficial effect of CR
resulting in a decreased lifespan (Boily, Seifert et al. 2008; Li, Xu et al. 2008). Therefore
the SIRT1 null mice may not be a good model for studying the role of SIRT1 in CR-
mediated life span extension. Instead, the heterozygous (HET) SIRT1
+/-
is considered to
be a better choice. To determine the role of SIRT1 in CR-mediated longevity in vivo, we
continued the CR study as shown by Ying et al previously (Li, Xu et al. 2008) and
collaborated with Dr. Rafael de Cabo to study the gene expression changes in SIRT1
mice under CR condition. Three to five-month-old mice from 3 different genotypes
(SIRT1
+/+
, SIRT1
+/-
, and SIRT1
-/-
)were single caged and fed with an ad libitum (AL)
diets or a diet providing 60% of the calories given to the AL group (CR). CR extended
78

both medium and maximum life span of SIRT1
+/-
and SIRT1
+/+
mice (Figure 3.1). The
medium life span in both genotype increased about 50% (Table 3.1). There was no
statistically significant difference in median lifespan between SIRT1
+/+
and SIRT1
+/-
mice
subjected to AL or CR (p=0.69/0.108) (Table 3.2), which suggests that reduced SIRT1
levels do not affect the longevity effects of CR. Notably, the maximum lifespan under CR
condition tended to be shortened in SIRT1
+/-
compared to SIRT1
+/+
mice (p=0.067).
However, no statistically significant difference was observed in median lifespan in the
SIRT1
-/-
mice subjected to AL or CR. The median and maximal lifespan were remarkably
longer in SIRT1
+/+
and SIRT1
+/-
compared to SIRT1
-/-
mice (p<0.01 for all the
comparison). Under CR, the female mice tended to live longer than male mice.
Table 3.1 SIRT1 mice summary
LS (all) LS (F) LS (M)
Diet N
(Censored
1
)
N
Female
N
Male
Median
LS, wk
Median
LS, wk
Median
LS, wk
SIRT1
+/+
AL 16 (7) 7(4) 9(3) 109 N/A 108
SIRT
+/-
AL 15 (6) 7(2) 8(4) 102 106 95
SIRT1
-/-
AL 14 (1) 7 7(1) 64 66 62
SIRT1
+/+
CR 13 9 4 165 175 151
SIRT1
+/-
CR 18 8 10 155.5 157 130
SIRT1
-/-
CR 12 7 5 83 93 73
(1)
Censored after passing the median survival for tissue collection.

79

Figure 3.1 Kaplan-Meier survival curves of SIRT1
+/+
,
+/-
and
-/-
mice in ad libitum (AL)
and CR condition.

Table 3.2 Pairwise comparison of each mice group

SIRT1
+/+
AL
SIRT1
+/-

AL SIRT1
-/-
AL
SIRT1
+/+

CR
SIRT1
+/-

CR
SIRT1
-/-

CR
SIRT1
+/+
AL NA 0.69 0.008 0.009 0.183 0.836
SIRT1
+/-

AL NA 0.035 0.006 0.077 0.023
SIRT1
-/-
AL
NA <.0001 <.0001 0.394
SIRT1
+/+

CR NA 0.108 <.0001
SIRT1
+/-

CR NA <.001
SIRT1
-/-

CR NA
Pairwise comparison of each mice group (Energy intake, genotype and gender) using Log
Rank test.

We also performed behavioral test on aged SIRT1
+/+
and SIRT1
+/-
mice (about 150 weeks)
under CR. For the Y-maze test, the higher score in total entries and % SAB, the better the
mice survived. It seems that SIRT1
+/-
mice performed better in Y maze test (Figure 3.2A).
However, due to the limited mice available, the results are not conclusive. For elevated
0 25 50 75 100 125 150 175 200
0
25
50
75
100
SIRT1
+/+
AL
SIRT1
+/-
AL
SIRT1
-/-
AL
SIRT1
+/+
CR
SIRT1
+/-
CR
SIRT1
-/-
CR
Weeks
Percent survival

P
ar
d
m
th
F
(E

lus Maze (E
rms might i
etect betwee
mice availabl
he SIRT1
+/-
m
igure 3.2 B
EPM) test.
EPM) test, th
indicate SIR
en the two g
le and in cer
mice might p
ehavioral te
he higher sc
RT1
+/-
mice w
groups (Figur
rtain health c
perform bett
ests of SIRT
core in % Ti
were less s
re 3.2B). Al
conditions to
ter in advanc
T1 mice. (A
ime spent o
tressed. No
lthough the t
o validate th
ced age.
A) Y-maze te
pen arm and
significant
two tests we
he results, it
est. (B) Elev
d % entries
difference
ere limited b
still implied

vated Plus M
80
open
were
by the
d that
Maze
81

Figure 3.3 CR increased SIRT1 expression is increased in response to CR. (A) SIRT1
mRNA levels were measured by qRT-PCR (n=3 for all groups). Error bars indicate SEM.
Star denotes statistically significance when compared to SIRT1
-/-
in both AL and CR
diets (B) SIRT1 protein levels were determined by Western blotting (n=2-3 for all
groups). Bars shown as mean+SEM. Figures come from Dr. Evi M. Mercken.

To determine if one copy of Sirt1 affects its expression, our collaborator Dr. Mercken
measured SIRT1 expression in the liver. A genotype-associated proportional decrease of
SIRT1 expression both at the mRNA and protein level under AL and CR dietary regimes
was observed (Figure 3.3A, B). In both SIRT1
+/+
and SIRT1
+/-
genotypes, the SIRT1
expression tended to increase in response to CR, in agreement with the increase in SIRT1
protein levels under CR for mice, rats and humans (Cohen, Miller et al. 2004; Nisoli,
Tonello et al. 2005; Civitarese, Carling et al. 2007). Although the protein levels seemed
to increase during, the SIRT1 +/- mice still showed lower protein levels compared to
AB
82

SIRT1
+/+
, in agreement with the decreased expression of genes regulated or affected by
Sirt1.
Table 3.3 Pathologies and abnormalities in old mice (> 24 months)
CR Gender Survival(weeks) Pathology (observations after 2 years)
SIRT1
+/+
F 175 Growth in the left eye. Growth in the cheek.
F 87
M 123
F 180
F 128
F 170 Lost fur
F 213
M 147 Left eye problem
M 165 Both eyes have infections, left eye is severely damaged
F 176
F 206
F 156
M 155

SIRT1
+/-
F 168 Back legs problem, can't move well
F 162
F 109
F 158 Infections in both eyes
M 103
M 107
M 180 Left eye is a little enlarged
M 78
F 156
F 182 Growth in the middle of both eyes
F 143
F 155 Growth in right ear (possibly tumor)
M 156
M 175 Both eyes are red (may bleeding)
M 83
M 160 Eye problem
M 153 Eye problem
M 82

Microarray analyses of gene expression in SIRT1 mice under both diets regimes were
performed by Dr. Mercken et al to study whether the levels of SirT1-dependent genes
were altered by CR in the liver. By comparing the gene expression profile SIRT1
+/+

83

versus SIRT1
+/-
, and SIRT1
-/-
, they identified 77 SIRT1-dependent genes (51 up-
regulated, 26 down-regulated, a total change of 19%) and 24 pathways (13 up-regulated,
11 down-regulated, a total change of 18%) that were altered in response to CR. Many of
these SIRT1-dependent genes were involved in inflammation and stress response
pathways, indicating some SIRT1 expression is important for the longevity extension
induced by CR.

3.4 Discussion
SIRT1 and its yeast homolog Sir2 have been suggested as potential mediators of some
anti-aging effects including life span extension of calorie restriction (CR), although their
roles in CR-dependent lifespan extension is not clear (Kaeberlein, Kirkland et al. 2004;
Fabrizio, Gattazzo et al. 2005; Guarente 2005; Lee, Wilson et al. 2006; Boily, Seifert et al.
2008). Our lab previously found that CR did not change the life span of mice lacking
both copies ofSIRT1, which had shorter life span compared to SIRT1
+/+
mice under an ad
libitum diet (Li, Xu et al. 2008). A similar survival study was done by Boily et al. They
reported that SIRT1 null mice were hyperrmetabolic with excessive lipid oxidation and
their liver mitochondria did not function efficiently. The 40% reduction in caloric intake
maintained the metabolic rate and increased the physical activity in normal mice but not
in SIRT1 mull mice (Boily, Seifert et al. 2008). In contrast to these metabolic
deficiencies in SIRT1 null mice, Li et al showed that SIRT1 inhibition increased
acetylation and decreased phosphorylation of IRS-2 and reduced Ras activation and
ERK1/2 phosphorylation, which in turn protected neurons against oxidative damage(Li,
84

Xu et al. 2008). Furthermore, they found, in disagreement with what Boily et al reported,
reduced oxidized proteins and lipids in the brain of old SIRT1 null mice. In addition,
behavioral tests performed in this study at advanced ages in the SIRT1
+/+
and SIRT1
+/-
mice suggest that SIRT1
+/-
mice perform normally. These results imply that SIRT1 plays
different roles in various cellular processes yet its role in CR needs further investigation.

It is clear that SIRT1 is essential for development and growth, central effects which
might outweigh the beneficial effects of CR. Our survival and microarray study took
advantage of heterozygote SIRT1
+/-
mice, which maintain a certain level of SIRT1
expression to partially restore the normal development as well as tissue and organ
functions and avoid premature death and dysfunction caused by physiological deficiency.
Our collaborator demonstrated that there was a dose-dependent change in SIRT1 mRNA
and protein levels. The survival curve (Figure 3.1) implied that there was no significant
difference in the median life span between SIRT1
+/+
and SIRT1
+/-
mice, suggesting that
SIRT1overexpression is not essential for normal life span. We also showed that CR
extended the life span of both SIRT1
+/+
and SIRT
+/-
mice by about 50% but that SIRT1
+/+

mice may reach a slightly longer maximum life span. CR tended to increase SIRT1
expression as well. Although SIRT1
+/-
mice were long-lived under CR, they displayed
more frequent defects/pathologies (44% incidence) including eye infections or redness,
abnormal growths and impaired motility compared to SIRT1
+/+
mice (31% incidence)
(Table 3.3), which was in agreement with previous studies demonstrating that SIRT1-
knockout mice had defective metabolism and diminished physiological improvement in
85

responses to CR (Chen, Steele et al. 2005; Boily, Seifert et al. 2008). However, the early
death of SIRT1 null mice may be independent of the CR effects because of their sickness.
These results indicate that some expression of SIRT1 is required for CR-mediated
beneficial effects to prolong life span.

It is known that SIRT1 regulates the expression or activity of a large set of essential
genes involved in various cellular processes. SIRT1 has been shown to have anti-aging
effects through the deacetylation-dependent activation of the stress response transcription
factor FOXO (Brunet, Sweeney et al. 2004). SIRT1 also plays an important regulatory
role in glucose metabolism by promoting insulin expression and secretion in pancreatic b-
cells (Kitamura, Kitamura et al. 2005; Moynihan, Grimm et al. 2005) and by regulating
PGC-1 α to induce mitochondrial fatty acid oxidation (Gerhart-Hines, Rodgers et al.
2007). In addition, SIRT1 was reported to deacetylate p53 and inhibit premature cellular
senescence (Langley, Pearson et al. 2002). The microarray analysis carried out by our
collaborators demonstrated that many SIRT1-dependent genes, altered in response to CR,
were involved in inflammation and stress response pathways, suggesting that some
SIRT1 is required for many beneficial effects of CR. In agreement with our data, another
group has shown that overexpression of SIRT1 improves mice healthspan but not lifespan
(Herranz, Munoz-Martin et al. 2010). We speculate that the only copy of SIRT1 partially
restored the expression of certain genes needed for CR effects. However, it is still unclear
which sets of SIRT1-affected genes or pathways are responsible for the CR-induced
beneficial health effects.
86

In summary, our survival and microarray study of SIRT1 heterozygote mice under caloric
restriction demonstrates that some SIRT1 expression but not its high expression is needed
for the beneficial longevity and health effect of CR. By contrast, maximum life span
extension by CR may need high levels of Sirt1. Larger studies with tissue specific SIRT1
KOs are needed to further elucidate the role of SIRT1 in CR-mediated effects on aging
and life span extension.
87

Chapter 4 Acetic Acid is a Leucine-derived Ketone
Body-like Metabolite Catabolized by tor/s6k Mutants to
Promote Chronological Longevity in S. cerevisiae
4.1 Introduction
The mechanisms underlying the aging process in mammals remain poorly understood.
The simple and short-lived yeast, worm and fruit fly have been studied extensively to
shed light on the genes and mechanisms that affect aging in mammals. The budding yeast
Saccharomyces cerevisiae, due to its short life cycle, the straightforward techniques
available to study it, a well mapped genome and the similarities between its genes and the
homologs/orthologs in higher eukaryotes, has served as the simplest among the major
model organisms to study aging. Yeast has been responsible for the identification of two
of the major aging regulating pathways: the Tor/S6K and the Ras/cAMP/PKA(Beck and
Hall 1999; Pedruzzi, Burckert et al. 2000; Fabrizio, Pozza et al. 2001; Fabrizio, Liou et al.
2003). S. cerevisiae has also provided some of the initial links between pro-aging
pathways and age-dependent genomic instability (McMurray and Gottschling 2004;
Madia, Gattazzo et al. 2008; Madia, Wei et al. 2009). The yeast chronological life span
(CLS), a measure of the chronological survival of a post-mitotic population of cells, and
the replicative life span (RLS), a measure of the replicative potential of an individual
mother cell, have been the two major methods to assess yeast aging (Kaeberlein,
Kirkland et al. 2005; Steffen, Kennedy et al. 2009). Mutation in either Tor1 or Sch9,
88

yeast orthologs of mammalian mTOR and S6K, and mutations in the Ras/cAMP/PKA
pathway extend both the CLS and RLS (Fabrizio, Pozza et al. 2001; Longo 2003;
Kaeberlein, Powers et al. 2005; Hlavata, Nachin et al. 2008) in part by activating stress
resistance serine threonine kinase Rim15 and in part by activating transcription factors
Msn2/4 and Gis1 (Pedruzzi, Burckert et al. 2000; Fabrizio, Liou et al. 2003; Pedruzzi,
Dubouloz et al. 2003; Wei, Fabrizio et al. 2008). CLS can also be extended by a
hormesis-dependent effect, initiated by exposure to reactive oxygen species (Pan,
Schroeder et al. 2011; Longo, Shadel et al. 2012). Because the Tor/Sch9 pathway is
known to be activated primarily by amino acids while the Ras/cAMP/PKA pathway by
glucose and considering the key role of Rim15, Msn2/4 and Gis1 in the life span
extension caused by calorie restriction (CR), these pathways represent the central pro-
growth and pro-aging network activated by nutrients (Rubio-Texeira, Van Zeebroeck et
al. 2010). In fact, yeast cells cannot divide if they lack both Ras1 and Ras2 or Tor1 and
Tor2 (Toda, Uno et al. 1985; Barbet, Schneider et al. 1996).

In agreement with the effect of restriction of glucose and other carbon sources in
extending the life span of a variety of organisms, both glucose and ethanol have been
shown to accelerate CLS (Fabrizio, Gattazzo et al. 2005; Wei, Fabrizio et al. 2009). We
had previously shown that either the removal of glucose and ethanol or an increase in pH
was sufficient to extend CLS (Fabrizio, Gattazzo et al. 2005). Burtner et al. proposed that
acetic acid and not ethanol is the primary factor promoting acidification, chronological
aging and apoptotic death, based in part on previous reports showing that high levels of
89

acetic acid promote apoptosis (Madeo, Herker et al. 2004; Burtner, Murakami et al. 2009).
They also proposed that CR increased chronological survival by reducing extracellular
acetic acid (AcAc) and that the longevity of sch9Δ and ras2Δ mutants was due to their
resistance to acetic acid. However, the alternative explanation proposed by our group is
that, at physiological levels, acetic acid does not play a central role in aging and promotes
aging simply by activating pro-aging pathways analogously to glucose and ethanol based
in part on the fact that mutations in TOR1, SCH9, and RAS2affect aging similarly
independently of the concentration of acetic acid in the medium(Longo, Shadel et al.
2012).

In this study, we investigated the relative role of glucose, ethanol, and acetic acid in
chronological aging and investigated how long-lived mutants utilize different carbon
sources and how this may affect longevity.

4.2 Materials and Methods
Yeast strains Majority of the Saccharomyces cerevisiae strains used in this study were
derived from DBY746 (MATα, leu2-3, 112, his3 ∆1, trp1-289, ura3-52, GAL+). Some
experiments were also performed in BY4741 (MATa, his3 ∆1, leu2 ∆0, met15 ∆0, ura3 ∆0) and
the derivatives to confirm the results obtained with DBY746 strains. One-step gene
replacement introduced previously was utilized to generate the knockout strains
(Longtine, McKenzie et al. 1998). The sch9 ∆, cyr1::mTn and ras2 ∆ mutants have been
described previously (Fabrizio, Pozza et al. 2001; Wei, Fabrizio et al. 2008). Prototrophic
90

strains were generated by transfer plasmids with URA, HIS, LEU or TPR into DBY746.
Strain with STRE-lacZ reporter gene were generated by transforming the plasmid pMM2
containing four tandem repeats of STRE motif from the HSP12 sequence (-221 to -241)
(Boy-Marcotte, Perrot et al. 1998)and integrating into the URA3 locus of wild type. The
plasmid pCDV454 containing LacZ reporter under the control of a 37bp SSA3-PDS
region (-206 to -170) (Pedruzzi, Burckert et al. 2000) was integrated into the URA3 locus
of wild-type cells to generate PDS-lacZ reporter gene strain. The reporter gene activities
have been described in previous study (Wei, Fabrizio et al. 2008).

Growth conditions Yeast cells were grown in SDC containing 2% or 0.5% glucose
supplemented with a 4-fold excess of the three essential amino acids tryptophan, leucine,
histidine and uracil to avoid possible lifespan modification due to auxotrophic
deficiencies of the strains. Normal standard SDC medium without excess of essential
amino acid were also utilized for comparison. Day3 wild type and day7 sch9 ∆ (day11 for
BY4741 sch9∆) expired culture were centrifuged and supernatants were filtered to
perform medium switch experiments.

Chronological life span assay Yeast chronological life span was measured as previously
described (Pedruzzi, Dubouloz et al. 2003). Overnight SDC culture was diluted (OD0.1)
into fresh SDC medium to a final volume of 10 ml (with 5:1 flask to culture volume) and
were maintained at 30°C with shaking (200 rpm).Twenty four hours later was considered
as day1. Every 48 hours, properly diluted culture was plated on to YPD plates and then
91

incubated at 30°C for 2-3 days. Chronological life span was monitored by measuring
colony-forming units (CFUs). Day3 CFUs was considered as the initial survival (100%)
and used to determine the age-dependent mortality. Mean life span was calculated from
curve fitting (one phase exponential decay) of the survival data with the statistical
software Prism (GraphPad Software). For medium switch experiments, cells were grown
in SDC for 3 days, washed twice with sterile distilled water, and suspended in MES
buffer (40mM, pH3.7 or pH6.0) with different carbon sources or other media as stated.
Stress resistance assay Check chapter2 materials and methods

LacZ reporter gene assay Check chapter2 materials and methods

Acetic acid and ethanol measurements Yeast chronological aging cultures were
centrifuged and supernatants were collected and frozen at −80°C. Acetic acid and ethanol
concentrations of the expired medium were determined by R-biopham acetic acid assay
and ethanol assay kit (Cat. 10 148 261 035 and 10 176 290 035). Measurements followed
the manufacture recommend protocol with appropriate dilutions of each sample.

Oxygen consumption assay Oxygen consumption was measured by taking 2 ml of
culture into a glass container with a magnetic stir bar to avoid cell precipitation in a 37
o
C
water bath using a Clark-type electrode. According to the manufacturer, we assumed that
the liquid culture contains the same amount of oxygen as water equilibrated with 21%
oxygen in 1 atmosphere pressure (5.02 μl/ml). The amount of oxygen consumed was done
92

by converting the recorded value to nano moles and was further normalized by CFU.
Data were collected (program automatically recorded every 5 seconds) until a trace of
straight line was obtained which suggested the consumption of oxygen had reached a
steady state.

Quantification of Glycogen and Trehalose To look at glycogen and trehalose
accumulation, 2.5-3ml cell culture were collected from indicated cultures at indicated
time points and washed with water then stored at -70
o
C before measurement. The cell
pellet dry weight was measured at the same time of collecting for normalization purpose.
Determination of glycogen and trehalose content was performed as previously described
by Parrou and Francois (Parrou and Francois 1997). First, cell pellets were resuspended
in 250ul of 0.25 M Na
2
CO
3
and incubated for 4 h at 95ºC with occasional stirring.
Subsequently, the suspension was neutralized to pH 5.2 by addition of 150ul of 1M acetic
acid and 600ul of 0.2 M NaAc (pH 5.2). Next, for the trehalose content determination,
half of the mixture was incubated overnight at 37ºC in the presence of trehalase (Sigma,
USA) (3mU) under constant agitation. For the glycogen content determination, the
second half was digested overnight at 57ºC with continuous shaking on a rotary shaker in
the presence of α-amyloglucosidase (Sigma, USA) (100ug). Finally, centrifuge the
mixture and collect the supernatant. Glucose released from trehalose and glycogen
digestion was quantified using the Glucose Assay Kit (R-biopham) following the
manufacturer’s protocol.

93

4.3 Results

Figure 4.1 Quantification of extracellular acetate and ethanol during chronological aging.
(A, B) Extracellular acetate concentrations of wild type, sch9 ∆, ras2 ∆, tor1 ∆ andcyr1::Tn
mutants in a DBY746 background on days 1, 3, 5 and 7. Data are presented as mean ±
SEM (n = 3–7). (C, D) Extracellular acetate and ethanol concentrations of wild type,
sch9 ∆, ras2 ∆ mutants in a BY4741 background (n=3-6).

To establish the relationship between cell metabolites (carbon sources) and chronological
life span (CLS), we measured the contents of ethanol and acetic acid in aging cells. Wild
type cells accumulated acetate during chronological aging whereas longevity mutation in
either TOR1 or SCH9, the yeast orthologs of mammalian pro-aging genes mTOR and
S6Kinase (S6K) (Harrison, Strong et al. 2009; Laplante and Sabatini 2012), resulted in
depletion of extracellular acetate after a small acetate peak at day3. These data indicate
that the Tor1-Sch9 pathway blocks the utilization of acetate. By contrast, mutations in
RAS2 and CYR1, the central components of the other yeast pro-aging pathway which also
2 4 6 8 0
0
25
50
75
100
WT (DBY746)
sch9Δ
ras2Δ
cyr1::Tn
tor1Δ
Day
Extracellular Acetate (mM)
0 2 4 6 8 10
0
50
100
150
200
WT(DBY746)
sch9Δ
ras2Δ
Days
Extracellular Ethanol (mM)
2 4 6 8 10 12 14 16 0
0
25
50
75
100
BY ras2Δ
WT(BY4741)
BY sch9Δ
Day
Extracellular Acetate (mM)
0 2 4 6 8 10 12 14
0
50
100
150
200
WT(BY4741)
BY sch9Δ
BY ras2Δ
Day
Extracellular Ethanol (mM)
A B
C D
94

promote aging in mammals (Zhu, Woods et al. 1998; Ferbeyre, de Stanchina et al. 2002;
Okumura, Takagi et al. 2003; Yan, Vatner et al. 2007; Yan, Park et al. 2012),
accumulated similar or even higher acetate level compared to wild type strain (Figure
4.1A). These results suggest that during the early stages of lifespan as extracellular
glucose and other nutrients become depleted, the absence of Tor/Sch9 signaling promotes
a switch to an alternative metabolic mode which utilizes acetic acid, analogously to the
use of the acetoacetic acid and β hydroxybutyrate ketone bodies by mammalian cells
during fasting (Hawkins, Mans et al. 1986; Cahill 2006).

As we had previously shown, the ethanol content remained relatively high in wild-type
cultures until day 9, ruling out the possibility that wild type cells die from starvation
(Figure 4.1B). However, we confirmed that ethanol was depleted from the sch9 ∆ cultures
by day 3(Figure 4.1B) (Fabrizio, Gattazzo et al. 2005). The ras2 ∆ mutant depleted
ethanol even faster and by day3 ethanol was almost non-detectable in the culture. Calorie
restriction (CR) is well-known to extend longevity in a wide range of organisms
(Weindruch 1985; McCay, Crowell et al. 1989; Masoro 2005; Kennedy, Steffen et al.
2007; Anderson and Weindruch 2010; Omodei and Fontana 2011). Thus, the removal of
ethanol may partially contribute to the life span extension effects of Sch9 or Ras2
deficiency. To test whether these effects of Tor/S6K(Sch9) and Ras/cAMP/PKA on
extracellular carbon sources are common characteristics of yeast cells, we also measured
ethanol and acetate production in the BY4741 genetic background, one of the most
widely studied yeast genetic background, and obtained essentially the same results
95

(Figure 4.1 C,D). However, early carbon source depletion may only contribute to lifespan
extension in these mutants since in ras2 Δ mutants acetic acid remained in the culture at
very high levels (Figure 4.1A) and the replenishment of carbon sources every 2 days did
not abolish the lifespan extension effects of sch9 Δ mutations (Figure 5E).

To better understand how glucose and ethanol as well as acetic acid affect chronological
lifespan (CLS), we switched day 3 cells to MES buffer containing glucose, ethanol or
acetic acid. As shown in Figure 4.1, the average extracellular acetate concentration
between day3 and day 7, the period in which over 50% of the cells dies, is approximately
50mM. The pH of the medium was adjusted to that reached by wild-type cells in the
standard CLS assay (3.7). The standard 2% glucose concentration or a 100mM acetic
acid concentration, which is twice as high as the physiological levels (Figure 4.3A),
caused the highest mortality in agreement with previous studies (Figure 4.2A) (Granot
and Snyder 1991; Ludovico, Sousa et al. 2001; Burtner, Murakami et al. 2009). By
contrast, a physiological concentration of ethanol (0.8%) or of acetic acid (50mM) using
the standard low aeration protocol (Longo, Shadel et al. 2012) shortened lifespan less
than glucose or 100mM acetic acid, suggesting that the effect of physiological levels of
acetic acid on chronological aging is similar to that of ethanol and less compared to that
caused by glucose (Nishikawa, Edelstein et al. 2000; Smith, McClure et al. 2007).
96

Figure 4.2 Carbon sources affect chronological aging and stress response. (A)
Chronological survival of wild type (DBY746) in different media. Day 3 cells were
washed and transfer to MES buffer with different carbon sources at the concentrations
shown in the parenthesis. All media were adjusted to the same pH as that of control cells
(buffer only). B. Stress resistance assay of wild type cells 24 hours after the switch to
various buffers. Cells were heated for 80min at 55 degrees or treated with 80mM H
2
O
2

for 30min. A representative experiment is shown. (C, D) LacZ reporter assay. Wild-type
cells integrated with STRE- or PDS-lacZ gene elements were switched to buffer
containing different carbon sources on day 3. The STRE-lacZ (C) and PDS-lacZ (D)
activities were measured 8 h and 24 h after the switch. Data shown are mean ± SEM (n =
3–4). (E and F) wild type (DBY746) cells integrated with STRE- or PDS-lacZ gene
elements were grown in 2% glucose SDC medium until day2, washed twice with distilled
sterile water and then separated and transferred to 40mM MES buffer (pH 6, pH 4)
supplemented with ethanol (0.8%) or acetic acid (50mM). The STRE-lacZ (E) and PDS-
lacZ (F) activities were measured 8 h and 24 h after the switch (n=3). Star denotes p<0.05,
double stars denote p<0.01. (G, H) Cells were grown in 2% glucose SDC medium until
day2. Then cells were equally split into two flasks and one culture was buffered with
0.1M MES and NaOH to pH 6. Samples were collected 8, 24 and 48 hours after buffering
(n=3). Star denotes p<0.05.
2 4 6 8
0
25
50
75
100
125
Buffer (pH 3.7)
Ethanol(0.8% )
Glucose (2% )
Acetic acid(10mM)
Acetic acid (50mM)
Acetic acid (100mM)
Day
Survival (%)
8 24
0.0
1.0
2.0
3.0
4.0
Hours after switch
STRE-LacZ act.(U/mg)
8 24
0.0
0.2
0.4
0.6
0.8
Buffer(pH3.7)
Glucose(2%)
Ethanol(0.8%)
Acetic acid(10mM)
Acetic acid(50mM)
Acetic acid(100mM)
Hours after switch
PDS-LacZ act.(U/mg)
4 8 24
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Hours
STRE-LacZ act.(U/mg)
4 8 24
0.0
0.1
0.2
0.3
0.4
0.5
0.6 pH 6
pH 6 Ethanol
pH 6 Acetate
pH 4
pH 4 Ethanol
pH 4 Acetate
Hours
PDS LacZ act.(U/mg)
8 24 48
0.0
1.0
2.0
3.0
4.0
5.0
Hours
STRE-LacZ act.(U/mg)
8 24 48
0.0
0.1
0.2
0.3
0.4
pH 3.7
pH 6

Hours
PDS-LacZ act. (U/mg)
A B
C D
E F
G H
Control H
2
O
2
(70mM)
Buffer (PH3.7)
Ethanol(0.8%)
Glucose(2%)
Acetic acid(10mM)
Acetic acid(50mM)
Acetic acid(100mM)
HS100’
97

To examine whether the carbon sources influence stress resistance, we challenged cells
with heat shock and oxidative stress (H
2
O
2
treatment for 30min) 24 hours after switching
them to the various carbon sources. At physiological concentrations, all the carbon
sources sensitized yeast cells to oxidative stress, but not heat stress. Notably, a low
concentration of acetic acid had little effect on stress resistance (Figure 4.2B).

Transcription factors Gis1 and Msn2/4 are required for enhanced stress responses and
lifespan extension in Tor-Sch9 and Ras/cAMP/PKA deficient cells, in part because of
their effects on increasing the expression of stress resistance genes containing PDS and
STRE elements in their promoter region. To further understand how the carbon sources
affect the stress response, we employed STRE- and PDS-driven LacZ reporter gene assay
to monitor the gene expression changes in response to different carbon sources. High
concentration of acetic acid (100mM) almost completely repressed the STRE- and PDS-
dependent transactivation, while physiological concentrations of AcAc (50mM) or 10
mM AcAc did not reduce the activation of these transcription factors (Figure 4.2C, D), in
agreement with the survival results (Figure 4.2A). Twenty-four hours after the switch,
cells in glucose medium showed significant reduction in PDS-driven gene expression in
agreement with the stress resistance results (Figure 4.2B, D). Using physiological levels
of acetic acid and ethanol, we did not observe any significant decrease in cellular LacZ
gene expression (Figure 4.2C, D).This effect of 100mM acetic acid on stress resistance
transcription factors, could explain the rapid cell death under super physiological levels
of AcAc. Based on the results we conclude that physiological concentrations of either
98

acetic acid or ethanol in combination with acidification (pH 3.7) similarly affect cell
protection and survival.

To determine the relative contribution of acidification to cellular survival and stress
sensitization we monitored the effect of pH on the activity of the Msn2/4 and Gis1 stress
resistance transcription factors. First, analogously to the experiment above, we switched
cells to MES buffer (pH 6.0 or 4.0) supplemented with either physiological level of
ethanol (0.8%) or acetate (50mM). Mild acidification (pH 4.0) did not significantly alter
PDS- or STRE-dependent transactivation. The increase in pH from 4 to 6 elevated STRE-
and PDS-dependent lacZ activity in the presence to acetic acid (Figure 4.2E, F),which
may explain why buffering acidic media to pH 6 extends longevity (Fabrizio, Gattazzo et
al. 2005; Burtner, Murakami et al. 2009; Kaeberlein 2010). In contrast, a pH increase
promoted PDS- but not STRE-dependent lacZ activity in ethanol medium (Figure 4.2F).
We also buffered the expired medium removed from day 3 wild type cultures to pH 6.0
and compared stress response with that in unbuffered expired medium. Cells in the pH 6
medium exhibited elevated PDS-LacZ activity, which reached an almost two-fold
increase compared to cells in the unbuffered pH 3.7 medium by 48 hours (Figure 4.2G).
However, these changes were not observed for STRE-activity (Figure 4.2H). These
results suggest that Gis1-dependent effects via the PDS elements, which are mostly
negatively regulated by the Tor-Sch9 signaling pathway, may be responsible for part of
the effects of medium acidification and ethanol or acetic acid on stress resistance and
CLS.
99

Figure 4.3 Technical procedures affected acetic acid accumulation. Wild type cells were
grown in 2% SDC under standard condition with limited aeration (aluminum caps) or
high aeration condition (loose plastic caps). Extracellular acetate and ethanol
concentrations were measured every other day. (C) The pH of the cultures during
chronological aging.

In order to reconcile these results related to acetic acid and aging and the proposal by the
Kaeberlein laboratory that acetic acid is a toxic molecule that causes acute toxicity
(Kaeberlein 2010; Longo, Shadel et al. 2012), we investigated whether differences in
technical procedures may be responsible. Using plastic caps that allow high aeration
instead of the aluminum foil caps that limit aeration used in our method, Burtner and
colleagues showed that ethanol was depleted rapidly and acetic acid levels rose, as
proposed by them (Burtner, Murakami et al. 2009). Therefore, we performed our viability
assay using both aluminum foil capped flasks (our standard method) and flask with lose
plastic cap (Kaeberlein laboratory’s method). The evaporation rate was much higher in
2 4 6 8 10 12
40
60
80
100
120
Day
Extracellular Acetate(mM)
0 2 4 6 8
0
50
100
150
200
WT(DBY746)
WT(DBY746) Air
Day
Extracellular Ethanol(mM)
0 2 4 6 8
3.0
3.5
4.0
4.5
WT (DBY746)
WT (DBY746) Air
Day
pH
A B
C
100

plastic cap covered flasks as the volume decreased by 50-70% by day 11 compared to a
15% decrease by day 11 in flasks with aluminum foil caps (data no shown). The higher
aeration also caused early depletion of ethanol and caused the levels of acetic acid to
reach a concentration close to 100mM (Figure 4.3A, B). We also measured the pH of the
expired media during both condition. As shown in figure 4.3C, wild type DBY746
cultures maintained a pH of about 3.5-3.8 throughout the life span and the pH remained
similar under higher aeration conditions. These results indicate that the methodology used
by Burtner et al caused early depletion of ethanol and increased the levels of acetic acid
to a toxic range in contrast to our method in which ethanol remains the major carbon
source and the concentration of acetic acid reached between day 3 and 5 is approximately
50mM. The lower levels of ethanol and higher levels of acetic acid using the high
aeration method adopted by the Kaeberlein laboratory, is likely to have been a major
factor influencing their results and conclusion that acetic acid reaches toxic levels during
yeast aging.

101

A

Figure 4.4 Genetic mutations affecting acetic acid levels, CLS, and pH. (A) The glucose
metabolism pathway leading to ethanol and acetate generation. (B) Chronological
survival of wild type, ady2 ∆, pdc6 ∆ and acs1 ∆mutants (n=5-9). (C) Extracellular acetic
acid concentration in the various mutants (n=3-7). (D) Chronological survival of wild
type cells (BY4741) and ald6 ∆ mutants (n=3). (E) Extracellular acetic acid concentration
in wild type cells (BY4741) and ald6 ∆ mutants (n=3). (F) The pH of expired media of
strains in DBY746 or BY4741 background during chronological aging. (G) Extracellular
ethanol concentrations of wild type, ady2 ∆, pdc6 ∆ and acs1 Δmutants.
Pyruvate
PDH
Acetyl-CoA
PDH bypass
Ald2,3,6(cyto)
Ald4,5(mt)
Acetaldehyde
Acetate
Pdc1,5,6
TCA cycle
Acs1,2
Ach1
Ady2
Ethanol
Adh1
Adh2
Glucose
0 2 4 6 8 10 12 14 16
0
25
50
75
100
125
150
Day
Survival (%)
1 3 5 7 0
0
20
40
60
80
WT(DBY746)
ady2Δ
acs1Δ
pdc6Δ
Day
Extracellular Acetate(mM)
2 6 10 14 18 22 26 30
0
25
50
75
100
125
Day
Survival(%)
0 2 4 6 8 10 12 14
0
10
20
30
WT(BY4741)
ald6Δ
Day
Extracellular Acetate(mM)
1 3 5 7 9 11 13 15 0
0
1
2
3
4
5
WT(DBY746)
ady2Δ
acs1Δ
pdc6Δ
WT(BY4741)
ald6Δ
Day
pH
2 4 6 8
0
25
50
75
100
125
150
175
WT(DBY746)
ady2Δ
acs1Δ
pdc6Δ
Day
Extracellular Acetate (mM)
B C
D E
F G
102

To investigate further the role of AcAc in S. cerevisiae chronological aging and
acidification, we explored the effect of several genes involved in acetic acid metabolism
on CLS and medium pH. ADY2 encodes an acetate transporter required for normal
sporulation (Figure 4.4A). PDC6codes for an isoform of pyruvate decarboxylase, which
decarboxylates pyruvate to acetaldehyde.Deletion of PDC6 is expected to affect
production of acetaldehyde from pyruvate, and, in turn, reduce acetate production. ACS1
is an acetyl-coA synthetase, whose deficiency can affect acetate production (Figure 4.4A).
Although mutants lacking Ady2, Pdc6 or Acs1 accumulated significantly or greatly
reduced levels of acetic acid, none of them displayed increased CLS, ethanol generation
or pH (Figure 4.4B, C, F, G), suggesting that the levels of acetic acid accumulated using
the standard CLS method used in our laboratory do not play a major role in either CLS
nor medium acidification.

Acetate is the product of the pyruvate dehydrogenase (PDH) bypass, which in
Saccharomyces cerevisiae requires acetaldehyde dehydrogenase (ACDH) (Figure 4.4A).
There are five members of the ACDH family: ALD2-6. Acetate is mainly produced by the
cytosolic PDH bypass via Ald6p (Saint-Prix, Bonquist et al. 2004). To further investigate
the role of acetic acid in CLS, we examined the effect of ald6 ∆ mutation in the BY4741
background and confirmed that it played an important role in acetate production as
almost no acetate was detectable during chronological aging (Figure 4.4E). However, the
chronological lifespan was not significantly affected by the ALD6 deletion confirming
our hypothesis that CLS under standard aeration conditions is not the result of acetic acid
103

toxicity (Figure 4.4D). In addition, the medium pH of the ald6 Δ mutant was not
remarkably different from wild type (Figure 4.4G), further supporting that acetic acid was
not an important player in medium acidification.

Figure 4.5 CR affected ethanol and acetic acid accumulation. (A) Extracellular acetic acid
and ethanol concentrations of aging cultures of cells grown in 0.5% glucose SDC. (B)
Expired culture pH during chronological aging. (C) Chronological life span of wild type
(DBY746) in medium switch between 0.5% glucose SDC and 2% glucose SDC expired
medium on day3 (D) Stress resistance assay were performed 24 hours after medium
switch.
WT(DBY746) 0.5% SDC
2 4 6 8 10 12
0
10
20
30
Acetate
Ethanol
Day
mM
in expired medium
WT(DBY746)
1 3 5 7 9 11
3
4
5
6
7
8
2% SDC
0.5% SDC
Day
pH
WT(DBY746)
2 6 10 14 18 22 26 30 34
0
25
50
75
100
125
2% Glc
0.5% Glc
2% ->0.5% med
0.5% -> 2% med
Day
Survival(%)
A B
C
D
2% Glc
2% -> 0.5% med
0.5% Glc
0.5% -> 2% med
2% Glc
2% -> 0.5% med
0.5% Glc
0.5% -> 2% med
Control HS120’ H2O2 (120mM)
104

In agreement with the results by other labs, caloric restriction by glucose limitation (SC
supplemented with 0.5% glucose) reduced the acetic acid accumulation and medium
acidification and also caused partial increase in pH (from 3.7 to 5.2) (Figure 4.5 A, B). At
the same time, the accumulation of ethanol was dramatically decreased suggesting that
glucose concentration plays an important role in the accumulation of both ethanol and
acetic acid (Figure 4.5A). We also performed a medium exchange between 2% SDC and
0.5% SDC growth condition on day 3 and tested stress resistance 24 hours after the
switch. We found that the switch to the medium from the 0.5% glucose culture was not
sufficient to elevate the resistance to different stresses especially oxidative stresses
(Figure 4.5D). The extended life span of cells growing in 0.5% glucose medium was
largely reversed by switching to the medium from the cultures grown in 2% glucose,
confirming that the levels of carbon sources in the medium play a central role in survival
(Figure 4.5C). However, the switch of cells originating from 2% glucose medium to that
of CR condition medium did not lead to a full life span extension, confirming the
presence of cell intrinsic beneficial changes caused by caloric restriction other than those
affecting medium composition.
105

Figure 4.6 Carbon sources and genomic stability. Day 3 cells were washed and
transferred to MES buffer with different carbon sources at the concentrations shown in
the parenthesis. All media were adjusted to the same pH as that of the control cells (pH
3.7). (A, B) Chronological life spans of wild type and sch9 Δ mutants in different media.
(C, D) Mutation frequency during aging in the CAN1 gene (measured as Can
r

mutants/10^6 cells, n =3).

We also investigated how carbon sources affect genomic stability during aging. Day3
wild type and sch9 ∆cells were switched to buffer (pH 3.7) supplemented with 2%
glucose, 0.8% ethanol or 50mM acetate (Figure 4.6). Although 2% glucose shortened the
life span of both wild type and sch9 Δmutants, it had small effects on mutation frequency
(Figure 4.6). Unlike what was observed for wild type cells, survival of sch9 ∆ mutants
was improved in 50mM acetate compared to that in 0.8% ethanol, indicating that sch9 ∆
mutants may be better adapted to high acetic acid concentrations (Figure 4.6B).
WT(DBY746)
2 4 6 8 10
0
25
50
75
100
125
Day
Survival (%)
WT(DBY746)
3 5 7 9
0
2
4
6
8
Day
Mutation Frequency
(CAN
r
mutants/10^6 cells)
sch9Δ
2 4 6 8 10 12 14 16
0
25
50
75
100
125
Buffer (pH 3.7)
Glucose (2% )
Ethanol(0.8% )
Acetic acid (50mM)
Day
Survival (%)
sch9Δ
3 5 7 11 15
0
2
4
6
Buffer (pH 3.7)
Glucose (2% )
Ethanol(0.8% )
Acetic acid (50mM)
Day
Mutation Frequency
(CAN
r
mutants/10^6 cells)
A B
C D
106

Although there was a trend for an effect of acetate on mutation frequency, the effect of
these carbon sources on lifespan is probably not related to genomic stability.
DBY746
2 4 6 8 10 12
0
25
50
75
100
125
Day
Extracellular Acetate(mM)
BY4741
2 4 6 8 10 12 14 16
0
10
20
30
40 4X(4AA)
1X(4AA)
4X(3AA) 1XLEU
Day
Extracellular Acetate(mM)
DBY746
2 4 6 8 10 12
0
25
50
75
100
125
Day
Extracellular Acetate(mM)
BY4741
2 4 6 8 10 12
0
50
100
150
200
250
4X(4AA)
1X(4AA)
4X(3AA) 1XLEU
Day
Extracellular Ethanol(mM)
DBY746
2 4 6 8 10 12
0
25
50
75
100
125
150
175
Day
Extracellular Ethanol(mM)
BY4741
2 4 6 8 10 12
3.0
3.5
4.0
4.5
4X(4AA)
1X(4AA)
4X(3AA) 1XLEU
Day
pH
A B
DBY746
2 4 6 8
0.0
0.5
1.0
1.5
2.0
2
6
10
Day
Extracellular Acetate
(mM/10^6) cells
DBY746
2 3 4 5 6 7 8
0
3
6
9
12
4X(4AA)
1X(4AA)
4X(3AA)1XLEU
Day
Extracellular Ethanol
(mM/10^6 cells)
C D
E F
G H

Figure 4.7 Branched chain amino acid leucine affects acetic acid generation. (A, B) Wild
type cells (DBY746 and BY4741) growing in 2% SDC with 4X or 1X HIS, URA, LEU,
TRP or 1X LEU with 4XHIS, URA,TRP. Extracellular acetate concentration during
chronological aging (n=3-5). (C, D)Extracellular ethanol concentration of DBY746 and
BY4741 wild type in different media during chronological aging (n=3-5). (E, F) Medium
pH in the cultures during chronological aging. (G, H) Extracellular acetate and ethanol
concentration normalized to cell numbers (n=3).
107

Acetic acid is very similar to mammalian ketone bodies and, analogously to ketone bodies, it is
produced following the depletion of extracellular nutrients. We hypothesized that acetic acid is a
ketone body-like metabolite that serves as an energy source during periods of starvation. In
mammalian cells, ketone bodies which include acetone, acetoacetic acid, and β-hydroxybutyric
acid are generated from fatty acids catabolism and also from leucine catabolism. Therefore, we
tested if leucine could affect acetic acid levels. The commonly used laboratory yeast strains
contain mutations in amino acid biosynthetic genes that facilitate genetic engineering, e.g. leu2,
his3 and ura3 (in the DBY746 and BY4741 genetic backgrounds, see material and methods).
Growth medium is commonly supplemented with excess (4x) nutrients (leucine, histidine and
uracil) to compensate for these genetic deficiencies (Longo, Ellerby et al. 1997). When we
incubated cells in SDC medium with only 1X of the amino acids/RNA above the extracellular
acetate concentration was strongly reduced while ethanol level was not affected (Figure 4.7 A, C).
The same results were obtained with 1X leucine supplementation with the other three components
still in a 4-fold excess. Notably, cell density during growth in 1x leucine reached only 25-30% of
that reached by growth in 4X leucine (normal growth condition) but the acetic acid production
was still greatly reduced when the effects were normalized per cell number (Figure 4.7G, H).
Similar results were obtained with the BY4741 genetic background (Figure 4.7B, D).
Furthermore, although there was a major difference in acetic acid production, the different levels
of leucine did not cause significant differences in the pH confirming that acetic acid is not a
major factor in medium acidification (Figure 4.7E, F).
108

Figure 4.8 Excessive leucine promoted acetic acid accumulation. The extracellular
acetate concentration of BY4741 wild type cells (A) and sch9 ∆ mutants (DBY746) (B)
grown in SDC media with 4X and 8X LEU (n=4).

We also asked if increasing leucine level would promote acetate production as well. We
grew cells in SDC with 8X leucine and found that acetate levels significantly elevated in
the BY4741 wild type strain but not in DBY746 wild type strain, which already displayed
relatively high levels of extracellular acetate (Figure 4.8A). To our surprise, 8X leucine
also promoted acetate accumulation in sch9 ∆ mutants (Figure 4.8B).

WT(BY4741)
2 4 6 8 10 12
0
25
50
75
100 4XLEU
8XLEU
Day
Extracellular Acetate (mM)
sch9Δ(DBY746)
2 4 6 8 10 12
0
25
50
75
100 4XLEU
8XLEU
Day
Extracellular Acetate (mM)
A B
109

Figure 4.9 Leucine catabolism and acetic acid generation. (A)Leucine catabolism
pathways in yeast and mammals. (B) Extracellular acetate concentration in cultures of
wild type cells and of mutants lacking BAT1 and BAT2genes (n=6), star denotes p<0.05.

Because these results suggested that leucine contributed to acetate generation, we looked
at the leucine catabolism pathway. Although leucine catabolism in mammals is well
understood, no well defined leucine catabolism pathway has been described for yeast.
Using mass spectrum and isotope-tagged carbon sources, a possible yeast leucine
metabolism pathway has been proposed but it has not been connected to ketone body-like
Acetoacetate Acetyl-CoA Fusel Alcohol Fusel Acid
Mammals Yeast
A
Leucine α- Ketoisocaproate Isovaleryl-CoA
branched-chain
amino acid
aminotransferase
Bat1 Bat2
branched-chain
alfa-keto acid
dehydrogenase
3 5 7
0
25
50
75
100
WT(DBY746)
bat1Δbat2Δ
Day
Extracellullar Acetate
(mM)
B
110

metabolites (Figure 4.9A) (Dickinson 2000). Bat1 and Bat2 are the mitochondria and
cytosolic branched-chain amino acid (BCAA) aminotransferases (Figure 4.9A). Deletion
of either of these two genes had little effect on acetate accumulation but double knockout
of Bat1 and Bat2 reduced extracellular acetate to very low levels during early
chronological aging (Figure 4.9B). These data confirm that leucine catabolism can lead to
acetate production and that acetate is a ketone body-like metabolite.

Figure 4.10 ACH1 was important for acetic acid utilization. (A) Chronological survival
of wild type, ach1 ∆, sch9 ∆ and sch9 ∆ach1 ∆ mutants (n=7). (B, C) Excellular acetic acid
and ethanol concentration with chronological aging (n=4).

The effect of inhibition of Tor-S6K signaling on longevity, originally described in S.
cerevisiae (Fabrizio, Pozza et al. 2001)and confirmed in worms, flies and mice, is one of
the most promising pharmacological intervention with the potential to extend healthy
lifespan in humans (Powers, Kaeberlein et al. 2006; Hansen, Taubert et al. 2007;
1 3 5 7 9 11 15
0
25
50
75
100
125
150
175
WT(DBY746)
sch9Δ
ach1Δ
sch9Δach1Δ
Day
Survival (%)
2 4 6 8 10 12 0
0
25
50
75
100
125
150
175
WT (DBY746)
sch9Δ
ach1Δ
sch9Δach1Δ
Day
Extracellular Acetate(mM)
1 3 5 7 9
0
50
100
150
200
250
WT(DBY746)
sch9Δ
ach1Δ
sch9Δach1Δ
Day
Extracellular Ethanol(mM)
A B
C
111

Harrison, Strong et al. 2009; Katewa and Kapahi 2011). To investigate the effect of
Tor/Sch9 deficiency on acetic acid depletion and the potential entry into an alternative
metabolic pathway we studied the role of ACH1, which codes for a CoA transferase, one
of the most important enzymes involved in acetate metabolism, that catalyzes the CoASH
transfer from succinyl-CoA to acetate but also has minor acetyl-CoA-hydrolase activity
(Figure 4.4A) (Buu, Chen et al. 2003; Fleck and Brock 2009). The ACH1 deletion
mutants and wild type cells displayed a similar mean lifespan although the maximum
lifespan of ach1 Δ was shorter (Figure 4.10A). The sch9 ∆ach1 ∆ double mutations caused
an almost complete reversal of the longevity effect of sch9 Δand caused a major increase
in ethanol and acetate accumulation compared to sch9 ∆ single mutants (Figure 4.10B, C).
The acetate in the sch9 Δach1 Δ mutant reached a concentration of over 100mM, which
could cause toxicity. However, the enhanced stress resistance in sch9 ∆ mutants was not
affected by deletion of ACH1 or impacted by the higher content of acetic acid in the
expired medium (data not shown). These results are consistent with the hypothesis that
the activation of an alternative acetic acid utilization pathway is dependent on ACH1 in
cells deficient in Tor-Sch9 activity, which must be activated to achieve lifespan extension.
112

Figure 4.11 Respiration and carbon sources utilization. (A) O
2
consumption of wild type
(DBY746) and sch9 Δ mutants during chronological aging. Cells were grown and
maintained in SDC medium for the entire study. Oxygen consumption was measured
every other day. Data are presented as mean + SE, (n=8-9). (B) O
2
consumption of wild
type (BY4741) and sch9 Δ mutants during chronological aging (n=2-5). (C, D) Wild type
and sch9 ∆ mutant cells grown in 2% SDC until day 3 and then washed 3 times and
switched to 40mM MES buffer(pH3.7) or buffer with physiological levels of
ethanol(0.8%) and acetic acid (50mM). Oxygen consumption were monitored 3 and 24
hours after the switch (N=4-8). (E, F)WT (DBY746) and sch9 Δ mutants were grown in
2% SDC and switched to the carbon source mix medium (40 mM MES buffer, pH 3.7:
with 50 mM acetic acid and 141 mM ethanol). The medium was changed on day 3 and
replaced every other day. Chronological survival and O
2
consumption were monitored for
the entire CLS study. Data are presented as mean + SE (n=7).

Previously, down-regulation of the Tor1-Sch9 signaling pathway was shown to induce an
early increase of respiration rates (Bonawitz, Chatenay-Lapointe et al. 2007; Pan and
0 2 4 6 8 10 12
0
50
100
150
200
sch9Δ
WT(DBY746)
Day
O
2
Consumption
(% of WT day1)
0 2 4 6 8 10 12
0
50
100
150
sch9Δ
WT(BY4741)
Days
O
2
consumption
% of WT day 1
WT(DBY746)
3 24
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Hours
O
2
consumption
(nmol/s/2*10^6 cells)
sch9Δ (DBY746)
3 24
0.0
0.5
1.0
1.5
2.0
Buffer (pH 3.7)
Buffer Ethanol
Buffer Acetate
Hours
O
2
consumption
(nmol/s/2*10^6 cells)
2 6 10 14 18 22 26
0
25
50
75
100
125
Day
Survival (%)
2 3 4 5 6 7 8 9 10 11
0
1
2
3
WT(DBY746)
sch9Δ
Day
O
2
Consumption
(nmol/2*10^6 cells)
A B
C D
E F
113

Shadel 2009; Wei, Fabrizio et al. 2009)in yeast as well as an early depletion in ethanol
and acetic acid during chronological aging (Figure 4.1A, B) (Fabrizio, Gattazzo et al.
2005). We hypothesized that inhibition of Tor-Sch9 signaling increases respiration by
promoting the catabolism of ethanol and acetic acid. Oxygen consumption measurements
revealed that mutation in SCH9 induced a high rate of respiration at early stages of
survival, which reached very low levels by day 7 (Figure 4.11A). In the BY4741 genetic
background, deletion of SCH9 exerted a similar but postponed effect on O
2
consumption
(Figure 4.11B). These metabolic changes matched the pattern of depletion of ethanol and
acetic acid during chronological aging. However, sch9 ∆mutants remained long-lived
when ethanol and acetic acid were replenished at physiological levels (141mM and
50mM) every other day during the CLS (Figure 4.11E, F), indicating that the effects of
Tor-Sch9 on CLS are not simply due to entry into a low metabolism phase. To examine
the effect of each carbon source on cellular respiration, we switched wild type and sch9 ∆
cells to MES buffer containing 0.8% ethanol, 50mM acetic acid on day 3 and measured
oxygen consumption 3 and 24 hours after the switch. Whereas ethanol promoted cellular
respiration, acetic acid, which was also rapidly depleted by sch9 ∆ mutants, did not
support high respiration even at points in which its levels were high (Figure 4.11C, D).
Therefore, deficiencies in the Tor-Sch9 pathways activate an alternative metabolic mode
that promotes rapid catabolism of ethanol, which promotes high respiratory rates and a
rapid catabolism of acetic acid. (Figure 4.11A, B was provided by Dr. Madia and data
was confirmed by the thesis author)
114

Figure 4.12 Electron transport activity is important for acetic acid utilization. (A, B)
Extracellular acetate and ethanol in the cultures of coq mutants on days 3 and 7 (n=3). (C,
D) WT, sch9 ∆, coq ∆ and sch9 ∆coq ∆ double mutants were switched to 40mM MES
buffer (pH 3.7) containing 25mM acetic acid or 0.8% ethanol. Acetate and ethanol
utilization was monitored. (E) The sch9 ∆ mutant is treated with NaCN on day1 then
every other day until day7. Acetic acid concentration in the expired medium was
monitored every other day.

To further investigate the role of respiration on survival and utilization of metabolites, we
treated wild type and sch9 ∆ mutant cells with NaCN (0.25mM), which is a respiration
3 7
0
20
40
60
80
Day
Extracellular Acetate (mM)
3 7
0
50
100
150
200 WT(DBY746)
sch9Δ
coq2Δ
coq4Δ
coq2Δsch9Δ
coq4Δsch9Δ
Day
Extracellular Ethanol (mM)
2 4 6 8 10 12
0
50
100
150
Day
% of initial Acetic Acid
2 4 6 8 10 12
0
25
50
75
100
125
WT(DBY746)
sch9Δ
coq2Δ
coq4Δ
sch9Δcoq2Δ
sch9Δcoq4Δ
Day
% of initial Ethanol
2 4 6 8 10 12
0
5
10
15
20
25
sch9Δ
sch9Δ NaCN
Day
Extracellular Acetate (mM)
A B
C D
E
115

inhibitor by blocking electron transport chain in mitochondria, on day 1 or day 3.
Treatement of sch9 ∆ mutants with NaCN every other day until day7 delayed the
utilization of acetic acid by sch9 ∆cells (Figure 4.12E). We also employed mutants with
impaired respiratory function. Coq genes catalyze ubiquinone (coenzyme Q) biosynthesis,
which serves to transport electrons between the respiratory enzyme complex I, II and III
in the mitochondrial inner membrane. Deletion of Coq genes causes respiration
deficiency and affects cell metabolism. We deleted the COQ2 or COQ4 genes in wild
type cells and sch9 ∆ mutants. These mutants exhibited impaired ethanol consumption and
low acetic acid accumulation during chronological aging (Figure 4.12A, B). However,
after switching cells to 40mM MES buffer (pH 3.7) with 25mM acetic acid or 0.8%
ethanol on day 3, neither ethanol or acetic acid were metabolized by these mutants
(Figure 4.12 C, D). These results indicate that acetic acid catabolism in sch9 ∆ cells
requires a fully functional respiratory chain, but does not necessarily results in oxygen
consumption in the absence of ethanol. Thus, Tor-Sch9deficiency activates an alternative
metabolic acetic acid catabolism mode that does not appear to require high oxygen
consumption.

116

Figure 4.13 Acetic acid and reserve carbon sources. (A, B) Trehalose and
glycogencontents were measured duringchronologicalaging. (C, D) The sch9 Δ
mutantcellswereswitched to MES buffer (pH 3.7) containing 25mM acetate or 0.8%
ethanol on day3. Trehalose and glycogenweremeasured.

We have shown that acetic acid depletion in sch9 ∆ mutant is ACH1 and electron transport
–dependent but does not promote high oxygen consumption. To determine whether acetic
acid may contribute to other cellular functions not related to energy production, we
measured stored nutrients. Storage carbohydrates, mainly glycogen and trehalose, serve
as energy sources in stationary phase and are important for cell survival. Mutants unable
to store or utilize the storage carbohydrates have significantly shortened CLS (Favre,
Aguilar et al. 2008). Respiration is important for the storage of carbohydrates as
respiratory-null strains have defective trehalose synthesis and are unable to store
glycogen (Filipak, Drebot et al. 1992; Yang, Chun et al. 1998; Enjalbert, Parrou et al.
2 4 6 8
0
5000
10000
15000
20000
Day
Glucose released from
trehalose digestion
(mg/g DW)
2 4 6 8
0
5000
10000
15000
sch9Δ
sch9Δ Ethanol
sch9Δ Acetate
Day
Glucose released from
glycogen digestion
(mg/g DW)
0 2 4 6 8
0
5000
10000
15000
20000
25000
Day
Glucose released from
trehalose digestion
(mg/g DW)
0 2 4 6 8
0
5000
10000
15000
20000
WT(DBY746)
sch9Δ
Day
Glucose released from
glycogen digestion
(mg/g DW)
A B
C D
117

2000). Therefore, we measured the glycogen and trehalose contents of cells during
chronological aging. The levels of trehalose and glycogen decreased with age in wild
type cells and the sch9 ∆ mutant. The sch9 ∆ mutant stored 2-fold or higher glycogen and
trehalose compared to wild type cells (Figure 4.13A, B). Ocampo et al also showed a
positive correlation between stored carbohydrates content and cell survival (Ocampo, Liu
et al. 2012). This implies that the high level of trehalose may contribute to cellular
protection in tor1∆ and sch9 ∆ mutants. Glycogen does not appear to be as important
since by day 5 wild type cells and sch9 ∆ reach similar levels of the reserve nutrient
(Figure 4.13B). We then treated cells with acetic acid or ethanol to determine if these
carbon sources can be used to promote the storage of reserve carbohydrates. Surprisingly,
ethanol and acetic acid supported the storage of different carbon sources (Figure4.13C,
D). In sch9 ∆ mutants, acetic acid increased the synthesis of trehalose while ethanol
promoted its utilization. In contrast, ethanol but not acetic acid increased glycogen
content (Figure 4.13C, D). Therefore, ethanol and acetic acid enter different metabolic
pathways leading, respectively, to respiration and glycogen storage or trehalose
accumulation.
118

Figure 4.14 A model for the role of the Tor-Sch9 pathway in preventing the switch of
cells to an alternative metabolic mode that promotes longevity. In wild type cells, ethanol
was generated from glucose metabolism and utilized by respiration. Acetic acid was
instead produced from glucose and leucine metabolism. In wild type cells, high
respiration and low trehalose levels during late survival are associated with a short
lifespan. In sch9 Δ mutant, acetic acid was depleted by mechanisms that required ACH1
and electron transport respiration and that promoted the storage of the protective
carbohydrate trehalose. In sch9 Δ mutant, ethanol was depleted by enhanced respiration
in early diauxic shift stage and promoted glycogen storage. These changes are required
for chronological life span extension.

4.4 Discussion
The effect of energy in accelerating aging is well established in organisms ranging from
bacteria to mice and other mammals. In fact, a 20-40 percent restriction in calories
Glucose Pyruvate Acetaldehyde
Ethanol
Acetic Acid Acetyl-CoA
Leucine
Respiration
BAT1
BAT2
Aging
WT
sch9 Δ
ADH2 ADH1
Glucose Pyruvate Acetaldehyde
Ethanol
Acetic Acid Acetyl-CoA
Respiration (early diauxic shift)
Trehalose
ADH2 ADH1
Hypometabolic (late diauxic shift)
Glycogen
?
?
Longevity
?
ACH1
119

generally obtained by equally lowering the different macronutrients extends life span and
reduces the incidence of a variety of diseases (Lin, Defossez et al. 2000; Lee, Wilson et al.
2006; Wei, Fabrizio et al. 2008; Colman, Anderson et al. 2009; Katewa and Kapahi 2010).
Because high levels of acetic acid have been shown to cause apoptosis in yeast (Ludovico,
Sousa et al. 2001), the discovery that acetic acid accumulates in cultures of non-dividing
yeast resulted in the hypothesis that it may promote toxicity independently of the aging
process (Burtner, Murakami et al. 2009; Kaeberlein 2010). Here we show that under the
high aeration conditions used by others, ethanol is depleted and acetic acid accumulates
and can reach the toxic range associated with acute cell death whereas under the standard
aeration conditions used in our laboratory ethanol and acetic acid represent the major
carbon sources in the medium but ethanol appears to be the more potent pro-aging
macronutrient. We also show that, analogously to the ketone bodies acetoacetic acid and
3-β hydroxybutyrate in mammals, in yeast acetic acid accumulates during the early
periods of starvation and can be generated from leucine catabolism. Whereas wild type
cells and long-lived Ras pathway deficient strains accumulated high levels of acetic acid,
Tor-Sch9 deficient mutants could rapidly catabolize it by an ACH1- and electron
transport-dependent process that was required for lifespan extension and resulted in the
accumulation of the protective reserve carbon trehalose.

Small differences in the methods used to measure lifespan, can in some cases result in
changes that can have potent effects on the rate of aging and cell death as well as the
interpretation of the results (Gems and Partridge 2012). We published that glucose and
120

ethanol promoted aging and that medium acidification accelerated this process (Fabrizio,
Gattazzo et al. 2005). We also previously showed that the calorie-restricted conditions
achieved by removing ethanol, were sufficient to extend CLS (Wei, Fabrizio et al.
2009)analogously to the effect of reduction of glucose levels on both RLS and CLS.
Burtner et al, on the contrary, proposed that it was the accumulation of acetic acid and not
ethanol that limited yeast aging and proposed that acetic acid may limit CLS not by
promoting aging but by causing acute toxicity (Burtner, Murakami et al. 2009;
Kaeberlein 2010). Here we explain this apparent differences by showing that the high
aeration generated by experimental conditions analogous to those adopted by Burtner et
al instead of the reduced aeration obtained with the aluminum foil caps used in our
system, causes an early depletion of ethanol and the accumulation of levels of acetic acid
associated with toxicity, as described by Burtner et al. Thus, to identify genes and
pathways that are in fact affecting aging and not other processes, it is important to use
multiple systems (Longo, Shadel et al. 2012). For example, in both the water incubation
and the solid plate systems to study chronological aging in yeast, acetic acid is either
absent or present at very low concentrations in the medium, which rules out a key and
general role for acetic acid in CLS regulation (Wei, Fabrizio et al. 2008).

Ketone bodies, including acetone, acetoacetic acid, and beta-hydroxybutyric acid, are the
by-products of fatty acids catabolism in the liver to serve as a form of energy. They are
the preferable energy source in the brain during fasting (Hasselbalch, Knudsen et al. 1994;
Ludovico, Sousa et al. 2001; Kodde, van der Stok et al. 2007). The level of ketone bodies,
121

normally very low in the plasma, increases when the blood glucose level is low, with 3- β
hydroxybutyrate serving as the most abundant ketone body, once glycogen becomes
depleted (Cahill 2006). Ketone bodies are produced from acetyl-CoA, but can also be
generated from branched-chain amino such as leucine. No ketone body has been
described for S. cerevisiae. In yeast, acetate is produced by glucose metabolism,
converted from acetaldehyde and acetyl-CoA, analogously to ketone bodies generation.
Here we describe a number of parallels between yeast acetic acid and mammalian ketone
bodies 1) they are generated at high levels during periods of external glucose deprivation
and glycogen depletion, 2) leucine catabolism promotesketone body accumulation in
mammals and acetate accumulation in yeast (Bixel and Hamprecht 1995), 3) as for
ketone bodies, acetate can be transformed into Acetyl-CoA which enters the TCA cycle
for energy generation. The leucine metabolism pathway in yeast is still elusive. The
leucine metabolism pathway in yeast is still elusive. A previous study of leucine
catabolism in yeast did not find acetate as a byproduct (Dickinson 2000). This is probably
due to the fact that the experiments were not performed when nutrients were depleted in
the medium.

Sch9 and Tor1 deficiencies have been shown to extend life span, increase stress
resistance and promote genomic stability in S. cerevisiae (Fabrizio, Pozza et al. 2001;
Fabrizio, Gattazzo et al. 2005; Madia, Wei et al. 2009). Here we report that deletion of
Sch9 causes acetate utilization in an ACH1 and electron transport chain dependent
manner. However, in sch9 Δ mutants AcAc did not increase respiration analogously to
122

ethanol, suggesting that Tor-S6K deficiency switches cells to a starvation response mode
in which cells are able to utilize a ketone body-like metabolite for effects other than
energy production. Although it is not clear whether acetic acid can result in ATP
generation, we show that it promotes the accumulation of the stress resistance and reserve
carbon source trehalose, probably by a mechanism that requires an intact electron
transport, as indicated by others (Filipak, Drebot et al. 1992). Enjalbert et al. also found
that petite cells lacking mitochondrial DNA stopped synthesize trehalose as soon as
exogenous glucose was consumed while wild type continued accumulating the
disaccharide (Enjalbert, Parrou et al. 2000). By contrast, we show that ethanol promoted
glycogen accumulation. A recent study suggested the respiration thresholds were crucial
for the extended CLS by CR and trehalose was sufficient to restore the CLS of respiration
deficient cell (Ocampo, Liu et al. 2012). In agreement with our results, an early increase
in respiration has been shown for Tor deficient mutants and has been proposed to be
required for life span extension (Bonawitz, Chatenay-Lapointe et al. 2007). Another
group also reported that elevated respiration is required for CLS extension in Sch9
deficient cells but not for RLS (Lavoie and Whiteway 2008). However, RLS extension by
CR is suggested to be dependent on increased respiration (Lin, Kaeberlein et al. 2002).
Here, we show that this early increase in respiration in Tor/Sch9 deficient cells is
followed by a decrease in respiration after ethanol and acetic acid are largely depleted.
However, carbon source depletion and hypometabolism may contribute to but do not
explain the effects of Tor/Sch9 deficiency on lifespan since tor1∆ and sch9 ∆ mutants
123

display extended lifespan when carbon sources and respiration are maintained high and
constant (Fig. 4.12C).

In summary, our study characterizes acetic acid as a ketone body like molecule to serve
as an energy source for cell survival and also promotes cell death in a dosage dependent
manner. Furthermore, our data points to an effect for Tor-Sch9 deficient mutants in
switching to a mode, which is essential for life span extension, that enhances metabolism
of the ketone body-like acetate through Ach1 and electron transport activity resulting in
the accumulation of the protective trehalose (Figure 4.14).

124

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Abstract (if available)

Abstract Increased dosage of Sir2, a conserved histone deacetylase, extends replicative life span in yeast, and possibly worms, and flies and protects mammals against certain diseases. Previous work in our lab has shown that it is the lack of Sir2 and not its overexpression that promotes resistance to different stresses and extends chronological lifespan when combined with calorie restriction (CR) and/or mutations in the Tor/Sch9 or Ras pathways in yeast. To identify genes and pathways that mimic the effects of CR on aging and cellular protection we explored the role of Sir2 in regulating stress response and examined the genetic interaction between Sir2 and stress resistance transcription factors, Msn2/4 and Gis1. Our results suggest that Msn2/4 and Gis1 are required for the enhanced stress resistance of sir2Δ mutant and the further extension of chronological lifespan of sir2Δ mutant under caloric restriction. In agreement with this result, the serine/theronine kinase Rim15, a positive regulator of Msn2/4 and Gis1, was implicated in Sir2 mediated cellular sensitization to stress. We also examined the role of Sir2 in genomic stability during chronological aging and proposed a potential mechanism for its action. Further study of Sir2 implicated its involvement in regulating Ras2 expression. These studies shed light on the investigation of Sir2's function in higher eukaryotes. ❧ The Sir2 homolog SIRT1 deacetylase, one of the best-characterized sirtuins in mammals, has been shown to mediate some of the beneficial effects of calorie restriction (CR). However, its role in CR-dependent lifespan extension still remains unknown and highly controversial. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild type mice on an ad libitum or a caloric resitriction diet. Here we demonstrated that the median lifespan of SIRT1+/- heterozygote mice in CR was identical to that observed in wild type mice but a higher frequency of pathologies was displayed in SIRT1+/- mice. Microarray gene expression analysis further revealed the possible relations between SIRT1 and CR. Our results suggest that some SIRT1 expression but not its high expression is required for the beneficial effect of CR in longevity and health. ❧ In mammals, several days of food deprivation lead to the accumulation of ketone bodies including acetoacetic acid in the blood. Here we show that as external glucose becomes depleted, S. cerevisiae convert leucine to the ketone body-like acetic acid, analogously to the conversion of leucine to acetoacetate in fasting mammals. Acetic acid promoted the activation of pro-aging pathways similarly to glucose and ethanol. Whereas wild type and ras2 mutant cells accumulated acetic acid, tor1 and sch9 mutants depleted it rapidly by a mechanism that required acetate CoA-transferase and that was essential for lifespan extension. In sch9mutants acetic acid was partly depleted by a mechanism that required oxidative phosphorylation and was utilized to promote the stress resistance carbon source trehalose generation. These results indicate that that the effect of Tor/S6K deficiency on CLS extension involves an alternative metabolic mode, in which acetic acid can be utilized for energy production and the storage of stress resistance carbon sources. These effects are reminiscent of those described for mammals in response to fasting and raise the possibility that the life span extending effect of inhibition of TOR/S6K in higher eukaryotes may also involve analogous metabolic switches.

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Creator Hu, Jia (author)

Core Title Studies of Sir2 and caloric restriction mimetic pathways in aging

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 02/20/2013

Defense Date 12/14/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag acetic ccid,caloric restriction,genomic instability,ketone bodies,OAI-PMH Harvest,Sir2,SIRT1,stress resistance

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Longo, Valter D. (committee chair), Tower, John G. (committee member), Zhang, Chao (committee member)

Creator Email huj@usc.edu,hujia05@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-221121

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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