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University of Southern California Dissertations and Theses
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UVRAG protects cells from UV-induced DNA damage by regulating global genomic nucleotide excision repair pathway
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UVRAG protects cells from UV-induced DNA damage by regulating global genomic nucleotide excision repair pathway
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1
UVRAG Protects Cells From UV-induced DNA Damage by
Regulating Global Genomic Nucleotide Excision Repair
Pathway
By
Qiaoxiu Wang
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2016
2
Acknowledgments
First of all, I would like to express my gratitude to my supervisor, Dr. Chengyu Liang, for
providing me the opportunity to do research in the lab. Dr. Chengyu Liang has shown me both
the excellent thinking ability and experiment skills in research, which not only helps me finish
my graduate research, but also provides me the priceless advice in my future career. Moreover,
her great support always helps me overcome difficulties and regain motivation. I also would like
to express my gratitude for my committee members Dr. Michael R. Stallcup and Dr. Zoltan A.
Tokes, for their valuable advice and help in my thesis.
Second, I would like to thank my senior colleague, Dr. Yongfei Yang for his great
support during my research. Dr. Yongfei Yang has trained me with great patience and excellent
skills, which improved both of my experimental skills and critical thinking.
Additionally, I want to thank my lovely lab members, Tian Zhang, Sara Pirooz, Yongfei
Yang, Doug O' Connell, Payam Khalilzadeh, Shanshan He and Xudong Zhang. We work
together like a harmonious family. Without their help, it would be not easy for me to complete
my research well and enjoy the science.
I also would like to thank our program director, Dr. Zoltan Tokes for his advice in my
study and Jim Lee, for his help.
Last but not least, I would like to thank my family, my parents, my brother and my
friends, for their love and support, which always give me the courage to overcome difficulties.
3
Table of Contents
Acknowledgments ......................................................................................................................... 2
Abstract .......................................................................................................................................... 5
Introduction ................................................................................................................................... 6
1.1 UV-induced DNA Damage .................................................................................................... 6
1.2 UV-induced Effect in Human Body ...................................................................................... 7
1.2.1 UV-induced Positive Effect ............................................................................................ 7
1.2.2 UV-induced Diseases ...................................................................................................... 7
1.3 Nucleotide Excision Repair Pathway ................................................................................... 10
1.3.1 Global Genomic Nucleotide Excision Pathway ............................................................ 10
1.3.2 Transcription Coupled Nucleotide Excision Repair Pathway ...................................... 12
1.3.3 Repairing Steps ............................................................................................................. 13
1.3.4 Base Excision Repair (BER) and DNA Mismatch Repair (MMR) Pathway ............... 15
1.4 UVRAG ............................................................................................................................... 16
1.5 Autophagy ............................................................................................................................ 17
Methods and Materials ............................................................................................................... 19
2.1 Cell Culture and Transfection .............................................................................................. 19
2.2 Plasmids ............................................................................................................................... 19
2.3 Antibodies, Fluorescent Dyes and Other Reagents .............................................................. 20
2.4 Immunofluorescence and ImageJ Analysis ......................................................................... 20
2.5 Immunoprecipitation and Immunoblotting .......................................................................... 21
2.6 Clonogenic Cell Survival Assay .......................................................................................... 22
2.7 Unscheduled DNA Synthesis (UDS) and Recovery of RNA Synthesis (RRS) Assays ...... 23
2.8 Gene Knockdown by shRNA ............................................................................................... 24
Results .......................................................................................................................................... 25
3.1 Essential Role of UVRAG in Protecting Cells from UV Damage ...................................... 25
3.2 UVRAG Is Required for UV-induced Photolesion Repair .................................................. 29
3.3 The Involvement of UVRAG In UV-induced Photolesion Repair is Independent of
Autophagy ............................................................................................................................ 33
3.4 UVRAG Regulates GG-NER Pathway ................................................................................ 36
Discussion .................................................................................................................................... 41
4
Reference ..................................................................................................................................... 44
5
Abstract
DNA damage caused by ultraviolet radiation (UVR) is the leading factor in skin cancer
development. Nucleotide excision repair (NER) is one of the main mechanisms responsible for
repairing UV-induced DNA damage caused by UV. UV-radiation Resistance Associated Gene
(UVRAG), though first identified as a UV-related gene, is generally known as an important
regulator in different cellular pathways such as autophagy and intracellular membrane
trafficking. Here, I identify the essential role of UVRAG in UV-induced photolesion repair. This
is mainly through the regulation of the global-genomic nucleotide excision repair pathway (GG-
NER). And this regulation is independent of autophagy. These findings demonstrate that
UVRAG is essential in UV-resistance through participation in the GG-NER pathway.
6
Introduction
1.1 UV-induced DNA damage
UVR is one of the most deleterious factors that threaten living organisms. There are three
different types of UVR: UV-A, UV-B and UV-C. UV-C (<280 nm) radiation cannot penetrate
the Earth's atmosphere because of the ozone layer. However, UV-B (280–315 nm) and UV-A
(315–400 nm) radiation can penetrate the atmosphere and cause side effects. UVR can cause
different types of damage to the body, but the most detrimental is to DNA (Sinha et al., 2002).
UVR induces two major types of mutagenic DNA lesions. One is cyclobutane–
pyrimidine dimers (CPDs) and the other is 6-4 photoproducts (6-4PPs) (Figure 1). Both types of
lesions are formed by covalent bonds. CPDs are formed when two adjacent pyrimidines form a
covalent bond at C5 and C6 position of the DNA carbon atoms. Alternatively, 6-4PPs are formed
when carbon atoms at C6 and C4 position form a covalent bond (Ravanat et al., 2001).
Figure 1 Chemical Structure of Dimeric Photoproducts Formed at TC (Douki et al., 2006)
Seventy five percent of UV-induced mutagenic DNA lesions were due to CPDs while
twenty five percent were due to of 6-4PPs. Both CPDs and 6-4PPs damage DNA structure by
7
distorting the DNA helix. However, 6-4PPs are more lethal to cells (Singh et al., 2000). CPDs
cause a bend or kink of 7–9° and 6–4PPs cause a bend or kinkc of 44°(Kim et al., 1995).
Additionally, after absorbing a second UV photon, 6-4PPs may become a Dewar valence isomer
by photoisomerisation (Douki & Cadet, 2001). Moreover, local parameters along the DNA
sequence affect the photochemistry of the two classes. For example, CPDs form in more flexible
DNA segment or single-stranded DNA such as at the edge of the TATA-box (Aboussekhra &
Thoma, 1999).
1.2 UV-induced Effect in Human Body
1.2.1 UV-induced Positive Effect
UVR exposure has both positive effects and negative effects on the body. Exposure to
UV light causes 7-dehydrocholesterol to be converted to previtamin D, which is then converted
into vitamin D in the kidney and liver. Vitamin D plays an essential role in the calcium and bone
metabolism. However, the spectrum of UV radiation responsible for vitamin D conversion also
plays an important role in the UV-induced diseases (Garibyan et al., 2010).
1.2.2 UV-induced Diseases
The most well known disease caused by UVR is skin cancer. In general, there are two
different types of skin cancer, melanoma and nonmelanoma. Nonmelanoma skin cancers
(NMSCs) include basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), which are
both strongly linked to UV exposure. Both BCC and SCC usually arise on skin commonly
exposed to sunlight (Armstrong & Kricker. 2001). One out of three diagnosed cancers in the US
are skin cancers and most skin cancers are BCC. The American Cancer Society estimated there
are 2.8 million new cases of skin cancer diagnosed in the US in 2010, making it the most
8
frequently diagnosed type of cancer. In 2012, 700,000 cases of skin cancers have been diagnosed
and 2 out of 10 of those cases were SCC.
Melanoma is the most dangerous type of skin cancer in the world. Although it only
occupies a small percentage of total skin cancer diagnosed, the mortality rate is very high. In
2012, The World Health Organization (WHO) reported 232,000 cases of melanoma were
diagnosed and 55000 deaths from melanoma globally. Many new drugs and new therapies have
been developed to treat melanoma, but most only extend the life of patients rather than cure
them. Furthermore, melanoma has a high chance of developing drug resistance. Many promising
drugs have become less effective in treating melanoma.
Melanoma arise from melanocytes, which play an important role in protecting skin from
UVR. Melanocytes occupy 1-2% of the epidermal skin cells and are located in the basal layer of
the skin. When UVR damages DNA in keratinocytes, tumor suppressor p53 is activated, leading
to the expression of pro-opiomelanocortin (POMC) gene. In turn, keratinocytes release alpha-
melanocyte-stimulating hormone (MSH) by keratinocytes (Cui et al., 2007). Alpha-MSH then
binds to melanocortin-1 receptor (MC1R) on melanocytes to triggering a cAMP cascade, which
in turn increases the transcription of microphthalmia-associated transcription factor (MITF)
(Levi et al., 2006). MITF initiates the synthesis of melanin from tyrosine. In melanocytes,
melanin is stored in the melanosome and secreted into keratinocytes (Slominski et al., 2004).
Once inside the keratinocytes, melanin localizes to the nucleus and protects the keratinocytes
from UVR-induced damage (Figure 2) (Meredith et al., 2004). Melanin absorbs the UVR and
expels it as heat by a chemical process called internal conversion.
9
Figure 2 Melanin protects Against UV-induced Damage (Garibyan et al., 2010)
While melanocytes play an important role in protecting cells from UV-induced damage,
they can also become cancerous. Presently, the relationship between melanoma and UVR is not
as clear as nonmelanoma skin cancers. Unlike nonmelanoma skin cancers, melanoma also occurs
in the other body parts exposed less to sunlight. Gene mutation, UVR and other unknown factors
may contribute together to the development of melanoma. For example, BRAF, an important
component in the RAS-RAF pathway, has been found to be mutated in more than 60% of
patients with melanoma (Yang et al., 2010). These mutations include BRAF V600E and BRAF
B600K. More than 90% of these mutations are found in BRAF V600E. However, how UVR and
gene mutation causes the melanoma still remains unknown. Some argue that gene mutation in
melanocytes make them more vulnerable to UV damage, which in turn causes more mutations in
critical genes or damages to repair pathways, leading to melanoma. However, others believe that
UV light first causes the critical gene mutation in melanocytes leading to melanoma. Presently,
10
evidence shows that different parts of UV spectrum contribute differently to melanoma
development (Gandini et al., 2005).
1.3 Nucleotide Excision Repair Pathway
Nucleotide excision repair pathway (NER) plays an important role in UV-induced DNA
damage (Sinha et al., 2002). The NER pathway mainly targets DNA helix-distorting lesions,
most of which are pyrimidine dimers, such as CPDs and 6-4 photoproducts. In general, the
process of NER repair is divided into four steps: recognition, verification, excision and gap
filling and ligation. Due to the differences in the recognition step: the NER pathway can be
divided into two different sub-pathways, global genomic NER (GG-NER) and transcription
coupled NER (TC-NER). As the name indicates, the GG-NER pathway scans the entire genome
to repair UV-induced DNA damage. On the contrary, the TC-NER pathway specifically repairs
the DNA strand in transcription state which is responsible for coding the protein. However, after
the recognition step, both GG-NER and TC-NER share the same downstream pathway to repair
the bulky, helix-distorting DNA lesions.
1.3.1 Global Genomic Nucleotide Excision Pathway
The GG-NER sub-pathway repairs damaged DNA in both the transcription and non-
transcription region, while the TC-NER pathway repairs damaged DNA only in the transcription
region. During the recognition step of the GG-NER pathway, the UV-DDB (ultraviolet radiation-
DNA damage-binding protein) complex and XPC recognizes damaged DNA. XPC forms a
complex with excision repair associated protein RAD23B and CETN2 (Costa et al., 2003). This
complex is responsible for recognizing DNA helix-distorting lesions with the associated protein.
XPC binds to the small single-stranded DNA (ssDNA) gap containing the damaged base pairs.
11
This complex also plays a critical role in recruiting TFIIH. TFIIH is composed of subunits that
function to unwind the damaged DNA, which is required for the damage verification and repair
step. Other factors that function in the recognition step are XPA and RPA. Without XPA, NER
complex cannot form or function (Volker et al., 2001). RPA is required for preceding incision
and in post-excision repair synthesis. Moreover, RPA enhances the binding of XPA with
damaged DNA by using six oligonucleotide/oligosaccharide (OB) folds (Patrick et al., 2002).
The binding affinity and specificity between DNA damage sites and XPC depends on the
types of DNA distortion. XPC has low affinity for the minor DNA helix distortions such as
CPDs. The UV–DDB (ultraviolet radiation–DNA damage-binding protein) complex, which is
composed of DDB1 (also known as XPE-binding factor) and the GG-NER-specific protein
DDB2, recognizes tiny distortions in the genome and facilitate the recruitment of XPC and the
downstream NER subunits to damage sites (Sugasawaet al., 2010). Thus, UV–DDB is an
important recognition protein complex in the GG-NER pathway (Cleaver et al., 2009). Cullin 4A
(Cul 4A)-Roc1 ubiquitin ligase complex (CRL4) is also recruited by UV-DDB1 (Marteijn et al.,
2014). The UV-DDB-Cul4A-Roc1 complex (referred to as CRL4
DDB2
) ubiquitinates histones and
recruits chromatin remodelers to UV damage sites (Duan & Smerdon, 2010).
HR23B (Rad23 homolog B) is an accessory protein which increases the binding affinity
of XPC and helix distroting (Sugasawa et al., 2008). However, it is unclear which factor
recognizes the damaged site first. Recent findings support the theory that XPC-RAD23B first
recognizes the damaged site before the XPA-RPA complex (Riedl et al., 2003).
GG-NER recognition must be strictly regulated in order to avoid fatal mistakes. The
ubiquitination step is critical in regulating recognition. The 8kDa ubiquitin protein can greatly
affect the stability, the conformational change and the localization of GG-NER subunits by the
12
covalent binding to target proteins. In the recognition step of the GG-NER pathway, XPC is the
main ubiquitination target and the CRL4
DDB2
complex is the key E3-ubiquitin ligase (Groisman
et al., 2003). When UV-DDB recognizes the helix-distorted DNA lesions and binds to CRL4.
Then DDB2 is polyubiquitinated and released, promoting the binding of XPC (Pines et al.,
2012). Meanwhile, activated CRL4 complex ubiquitinates XPC and increases its binding affinity
for the damaged DNA site. XPC undergoes sumoylation upon binding to the damaged DNA site,
leading to the recruitment of E3 ubiquitin ligase RING finger protein 111 (RNF111) (Wang et
al., 2005). RNF111 polyubiquitinates XPC at Lys63 and prepares for the following repair steps.
1.3.2 Transcription Coupled Nucleotide Excision Pathway
Unlike GG-NER, the TC-NER pathway does not require UV-DDB1 and XPC-RAD23B
to recognize DNA lesions. Instead, RNA polymerase II (RNAPII) probes the DNA for lesions.
RNAPII is arrested at the lesion activating cockayne syndrome A (CSA) and cockayne syndrome
B (CSB) (Fousteri & Mullenders. 2008), which results in cell apoptosis during DNA replication
(Ljungman et al., 1996). CSA is a complex that contains WD-40 motif, which is known for
protein-protein interaction. CSA interacts with the UV-DDB-Cul4A-Roc1 complex involved in
GG-NER recognition (Groisman et al., 2003). DDB-Cul4A-Roc1 complex is positively regulated
by the covalent attachment of Nedd8 to Cul4A (Bennett et al., 2010). CSB is an ATP-dependent
chromatin remodeler. CBS loosely binds to RNAPII and stimulates transcription. The binding
becomes strong when RNAPII is arrest (Citterio et al., 2000). When exposed to UVR, CSB
activates transcription factors for UV-repair related genes (Proietti-De-Santis et al., 2006).
Several TC-NER specific proteins such as ubiquitin-specific-processing protease 7 (USP7) and
XPA-binding protein 2 (XAB2) are involved in this process (Fousteri et al., 2006).
13
Ubiquitin plays an important role in TC-NER pathway regulation. CSA is the main target
of the CRL E3 ubiquitin ligase is CSA. CSA may be ubiquitinated, which in turn leads to its
degradation. When probing DNA damage sites, UVSSA activates eubiquitinating enzyme USP7
to TC-NER complexes, which inhibits CSA function and prevents CSB degradation. The stable
CSB and UVSSA together recruit and activate downstream NER steps with the ubiquitin-binding
domain (Zhang et al., 2012). Polyubiquitylation and degradation also occur in RNAPII
(Nakazawa et al., 2012). If DNA lesions cannot be repaired by TC-NER pathway, polymerase
arrests at the damage site, which in turn inhibits gene transcription. The removal of RNAPII may
not only preserve the mRNA, but also prevents cellular apoptosis.
1.3.3 Repairing Steps
After the GG-NER and TC-NER recognition sub-pathways, the TFIIH complex and other
related genes are recruited to the damage site. The CAK (CDK-activating kinase) component of
this complex dissociates and activates the helicase activity of TFIIH. XPD from the complex
TFIIH and XPA unwinds the double strand DNA in the direction of 5′–3 (Compe et al., 2012).
XPA binds an exposed 30 nucleotide segment on the single strand DNA. XPA binding act as a
second recognition signal for the damaged site. Meanwhile, RPA is recruited to cover
undamaged DNA (Orelli et al., 2010). The binding of XPA and RPA forms a so-called pre-
incision complex, which helps to fully extend the DNA and keep it stable (Fagbemi et al., 2011).
XPA also recruits two structure-specific endonucleases: XPG and XPF-ERCC1. Under the
induction of XPA, XPG is activated to cut the 3′ damaged end of the lesion, while XPF-ERCC1
is responsible for cutting the 5’ end of the same lesion. The DNA polymerase is recruited and
uses the undamaged site as the template to synthesize a new DNA strand. Once the synthesis is
completed, DNA ligase I or DNA ligase III is recruited to fill the gap.
14
Figure 3 The NER Pathway Mechanism (Marteijn et al., 2014)
15
1.3.4 Base Excision Repair (BER) and DNA Mismatch Repair (MMR)
Pathway
In addition to the NER pathway, there are two different excision pathways to repair the
DNA damage: base excision repair (BER), and DNA mismatch repair (MMR). The BER
pathway mainly repairs the small and non-helix-distorted base lesions, such as oxidative bases,
alkylation bases, deamination bases, sites of base loss, uracil and single-strand breaks (Zharkov.
2008). One example is 8-oxo- 7,8-dihydroguanine (8-oxoGua), which leads the G/C base pair to
become T/A during replication resulting in DNA damage (Shimizu et al., 2003). The BER
pathway is activated by glycosylase, which recognizes DNA damage sites. This site is known as
AP site (apurinic/apyrimidinic site) and removed by AP endonuclease. The resulting DNA
damage site can be repaired by either short patch (one single base) or long patch (2 to 9 bases)
(Wiederhold et al., 2004).
Unlike the BER pathway, MMR focuses on post-replication, which is also defined as
non-Watson–Crick base pairs. MMR repairs mis-incorporated bases that escape the proofreading
mechanism during the DNA replication. This includes the insertion and deletion loops that are
the result of the polymerase slippage during the DNA replication (Jiricny. 2013). For example, a
G/T or A/C mis-pairing of bases may be induced because of tautomerization of bases during the
cell cycle. MMR initially recognizes the parental strand and the daughter strand, then localizes to
the mis-paired site. After the recognition step, the mismatch single bases are excised, leaving a
gap. During the repair synthesis step, a new single strand DNA is created to fill the gap (Fukui.
2010). The repaired target bases can be between a few bases to thousands of bases.
16
Figure 4 Types of DNA Damage and Repair Mechanisms (Mathews. DNA Repair of
Cancer Stem Cells )
1.4 UVRAG
The ultraviolet (UV) radiation resistance-associated gene (UVRAG) was first identified
in agenetic screen to partially complement UV sensitivity of xeroderma pigmentosum (XP),
which is involved in UV sensitivity (Liang et al., 2006). There are four major domains in
UVRAG: a proline-rich (PR) domain, a lipid-binding C2 domain, a coiled-coil domain (CCD),
and a C-terminal domain and each of them contribute to the multifunction of UVRAG (Liang et
al., 2006).
UVRAG is generally known as a crucial autophagy protein because it regulates different
stages of the autophagy pathway (Liang et al., 2006). We found that the C-terminus of UVRAG
17
helps to stabilize the centrosome and regulates DNA-dependent protein kinase in double strand
DNA damage repair (Zhao et al., 2012). UVRAG also functions as a tumor suppressor and as a
regulator of apoptosis upon chemotherapy treatment (Liang et al., 2008; Yin et al., 2011; S. He,
O'Connell, Zhang, Yang, & Liang, 2014).
Our lab recently found that two conserved residues (Lys 78 and Arg 82), located in
UVRAG C2 domain, have critical roles in interaction with PI3P. This interaction not only
recruits UVRAG, but also serves as the scaffold for the RINT1-ER-tether to mediate retrograde
transportation from the Golgi to the ER (S. He et al., 2013).
Nevertheless, none of these previously identified mechanisms of UVRAG identified
previously explain its relationship with UV-induced resistance mechanism. Here, we identify the
relationship between UVRAG and UV resistance mechanism.
1.5 Autophagy
Autophagy comes from the Greek root “auto”(self) and “phagy” (eating), and it means
self-eating. In general, autophagy (also known as macroautophagy) is a lysosome-dependent
degradation process of cytoplasmic materials. This intracellular degradation system maintains
homeostasis and prevents cell death and when disrupted, plays an important role in
pathophysiological condition (Ravikumar et al., 2010).
The general process of autophagy includes several steps. The first step is the
sequestration of modified, unnecessary, or damaged cellular components and the formation of the
autophagosome. Then the cargo is transported to lysosomes for degradation, and utilization of
degradation products are utilized for energy regeneration. However, in contrast to the ubiquitin–
18
proteasome system, which only recognizes, recycles and degrades ubiquitinated proteins,
autophagy undergoes both nonselective and selective degradation processes (Mizushima. 2007).
Mounting evidence indicate that autophagy is involved in numerous biological processes
such as starvation, cellular component clearance, tumorigenesis, cell death, and age-related
amyloid accumulation and development. For example, the deletion of autophagy-related gene 7
(ATG7) was found to extend the animal survival rate in melanoma (Xie et al., 2015). Autophagy
is implicated in neurodegenerative diseases by contributing to amyloids plaques. Beclin 1, an
autophagy-effector, causes promotion of amyloid β accumulation in mice and is found to have
reduced expression at the beginning stage of Alzheimer disease (Pickford et al., 2008).
19
Methods and Materials
2.1 Cell Culture and Transfection
HeLa, HEK293T, A375, and immortalized MEF (iMEF) cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
(FBS; Invitrogen), 2 mM L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco-BRL).
DDB2 (GM02415), XPC (GM00677), XPA (GM04312; immortalized) and CSA (GM01856)
deficiency skin fibroblasts were obtained from Coriell Institute (New Jersey, USA) and cultured
in Eagles Minimum Essential Medium supplemented with 10% fetal bovine serum, 2 mM L-
glutamine and 1% penicillin-streptomycin. All the cells have been cultured in an incubator with
the atmosphere of 5% CO2 and 37°C. Transfections were performed with Calcium Phosphate
Transfection Kits (Clontech) or Lipofectamine 2000 (Invitrogen), following their manufacturer’s
instructions.
2.2 Plasmids
The cDNA of the full-length UVRAG was used as the template for the Flag- or HA-
tagged wild-type (wt) UVRAG and they were cloned into Kpn I/Not I sites of the
pcDNA5/FRT/TO vector (Invitrogen, USA). The cDNA of the UVRAG were used as the
template for the construction of pEF/puro-Flag-UVRAG wild-type and mutants, and they were
cloned into the Afl II/Not I sites of the pEF/puro-Flag vector. The shRNA-resistant UVRAG
were generated by using the Site-Directed Mutagenesis Kit (Clontech) and then cloned in frame
with a Flag tag into pcDNA5/FRT/TO or pBabe-neo vectors. All shRNA plasmids were
purchased from Open Biosystem. All constructs were confirmed by sequencing using an ABI
PRISM 377 automatic DNA sequencer (Applied Biosystems, Foster City, CA).
20
2.3 Antibodies, Fluorescent Dyes and Other Reagents
The following antibodies were used in this study: UVRAG (U7058, Sigma-Aldrich) at
1:1000; UVRAG (SAB4200005, clone UVRAG-11, Sigma-Aldrich) at 1:200; actin (sc-47778,
clone C4, Santa Cruz) at 1:10000; CPD (TDM-2, CosmoBio) at 1:1500; Cul4A (ab72548,
Abcam) at 1:100; DDB1 (ab109027 Abcam) at 1:100; Roc1 (ab133565, Abcam) at 1:100; DDB2
(ab181136, Abcam) at 1:100; XPC (ab155025, Abcam) at 1:100; XPC (ab6264, Abcam) at
1:100; XPB (ab27317, Abcam) at 1:100; Flag (F3165, clone M2; SigmaAldrich) at 1:2,000; Flag
(F2555, Sigma-Aldrich) at 1:2,000; HA (PRB-101P, Covance) at 1:1000, HA (MMS-101P, clone
16B-12, Covance) at 1:2,000; myc (PRB-150P, Covance) at 1:1000; Biotin-Flag (F9291, Sigma);
Biotin-IgG (B7264, Sigma); HRP-labelled or fluorescently labelled secondary antibody
conjugates, purchased from Molecular Probes (Invitrogen). Unless otherwise stated, all
chemicals were purchased from Sigma.
2.4 Immunofluorescence and ImageJ Analysis
Immunofluorescence microscopy was carried out as described previously (Liang et al.,
2008). In general, cells plated on coverslips were fixed with 4% paraformaldehyde (30 min at
room temperature). After fixation, cells were permeabilized with 0.1% Triton X-100 for 7 min
and blocked with 10% goat serum (Gibco) with PBS for 1 hr. Primary antibody staining was
carried out using antiserum or purified antibody in 1% goat serum for 2 hr at room temperature
or overnight at 4ºC. Cells were then extensively washed with phosphate-buffered saline (PBS)
and incubated with Alexa 488-, or Alexa 568-conjugated secondary antibodies in 1% goat serum
for 1 h, followed by DAPI (4’, 6’-diamidino-2-phenylindole) staining for 15 min. Cells were
mounted using Vectashield (Vector Laboratories, Inc.). Confocal images were acquired using a
Nikon Eclipse C1 laser-scanning microscope (Nikon, PA) fitted with a 60X Nikon objective (PL
21
APO, 1.4NA) and Nikon image software. All experiments were independently repeated several
times.
ImageJ can be used to compare the density of bands on western blot or
immunofluorescence. According to the instructions, images were converted to grayscale. A
rectangle was drawn around the target area. After the rectangle has been set in place, a profile
plot of each target area was created. Each profile plot represents the relative density of the
contents of the rectangle over each target area, and the baseline of the profile plot peak was
closed off in order to exclude background interference. Peak was highlighted, which labeled each
peak with its size. The final result was shown by a percentage of the total size of all of the
highlighted peaks and value can be moved to a spreadsheet program for further comparision.
2.5 Immunoprecipitation and Immunoblotting
For immunoprecipitation, cells were lysed in 2% NP40 or Triton X-100 lysis buffer (25
mM Tris at pH 7.5, 300 mM NaCl, 1 mM EDTA and 2% NP40 or 2% Triton X-100),
supplemented with a complete protease inhibitor cocktail (Roche) and Micrococcal Nuclease
(NEB M0247), followed by freeze-thaw and sonication (amplitude 15%, process time 10s, push-
on time 5s, and push-off time 1s). Protein A/G agarose beads were used for pre-clearing for 1 h
at 4°C, and whole-cell lysates (WCL) were maintained for immunoprecipitation (IP) with the
indicated antibodies. Generally, 1-4 µg commercial antibody was added to 1 ml WCL, then
WCL was incubated at 4°C overnight for 8-12 hr. In the second day, addition of protein A/G
agarose beads were added into the WCL and incubation was continued for another 2 hr.
Immunoprecipitates were extensively washed with NP40 lysis buffer and eluted with SDS–
PAGE loading buffer by boiling for 5 min.
22
For immunoblotting, samples were run on a 6%-15% SDS-PAGE gel, generally for
30min at 60V and another 60min for 120v, and transferred to a PVDF membrane (BioRad) with
150mA per membrane for 90min. Membranes were blocked with 5% non-fat milk at room
temperature for 1 hr, and probed with the indicated antibodies at 4°C overnight. The membrane
was washed with PBST for three times, 15 minutes per time. THorseradish peroxidase (HRP)-
conjugated goat secondary antibodies were used (1:5,000, Invitrogen) and. immunodetection was
achieved with the Hyglo chemiluminescence reagent (Denville Scientific), and detected by a Fuji
ECL machine (LAS-3000).
2.6 Clonogenic Cell Survival Assay
A clonogenic survival assay was performed as described previously (Franken et al.,
2006). Briefly, cells were seeded in 60 mm dishes. At 70-80% confluent, cells were treated with
UV-C (294 nm) or DNA-damage inducing chemicals (24 hr exposure). After the exposure or
drug treatment, cells were allowed to recover for 24 hours. Then cells were trypsinized, counted,
and re-plated in appropriate dilutions for colony formation. After 10 to 14 days of incubation,
colonies were fixed and stained in a mixture of 6% glutaraldehyde (Amresco) and 0.5% crystal
violet, carefully rinsed with tap water, and dried at room temperature. Plating efficiency (PE) is
determined for each individual cell line as described (Franken et al., 2006) and the surviving
fraction (SF) is calculated based on the number of colonies that arise after treatment, expressed
as PE. Each experiment was repeated three times.
23
2.7 Unscheduled DNA synthesis (UDS) and Recovery of RNA
synthesis (RRS) Assays
UDS detection was performed using a Click-iT DNA AlexaFluor Imaging kit (Life
technologies), according to the manufacturer’s instructions. Briefly, after global irradiation (20
J/m2), cells on coverslips were incubated for 4h with 5 µM 5-ethynyl-2’-deoxyuridine (EdU),
then washed with PBS, fixed and permeabilized before incubation for 30 min with the Click-iT
reaction cocktail containing AlexaFluor Azide 488. After washing, the coverslips were mounted
with mounting medium (Vectashield, Vector Labs, CA). Cell images were analyzed as for the
RRS assay (see below). For each sample, at least 200 nuclei (non-S-phase) were analyzed per
condition of three independent experiments. Of note, non S-phase cells can be easily
differentiated from strong signals from scheduled DNA synthesis in S-phase cells.
For RRS, RNA detection was performed using a Click-iT RNA AlexaFluor Imaging kit
(Life technologies), according to the manufacturer’s instructions. Briefly, cells were UV-C
irradiated (10 J/m2) and incubated for 5 min (as a reference to show transcription is inhibited by
UV irradiation) or for 4 hr at 37°C, followed by 2 hr incubation with 100 µM 5-ethynyl uridine
(EU). Cells were then fixed and permeabilized in 4% formaldehyde and 0.5% Triton X-100 in
PBS, and after washing with PBS, incubated for 30 min with the Click-iT reaction cocktail
containing AlexaFluor Azide 488. Cells were then washed with PBST (0.05% Tween-20), and
the coverslips were mounted with mounting medium (Vectashield, Vector Labs, CA). Images of
the cells were obtained with a Nikon Eclipse C1 confocal microscope, and the average
fluorescence intensity per nucleus was quantified by NIS-Elements software and normalized to
the mock-treated cells. For each sample, at least 200 nuclei were analyzed from three
independent experiments.
24
2.8 Gene Knockdown by shRNA
All shRNAs were purchased from OpenBiosystem. The sequence targeting human
UVRAG is: 5’-ACGGAACATTGTTAATAGAA-3’
25
Results
3.1 Essential Role of UVRAG in Protecting Cells from UV Damage
As mentioned previously, UVRAG was first identified in Xeroderma Pigmentosum (XP)
to partially complement UV sensitivity (Perelman et al., 1997). However, previous studies on
UVRAG were mainly focused on its multi-functions such as membrane trafficking and
autophagy, rather than UV sensitivity. Hence, we explored whether UVRAG had an essential
role in protecting cells from UV damage.
In order to identify whether UVRAG protects cells from UV damage, we first performed
clonogenic cell survival assay. We chose A375 human melanoma cells and established two
different kinds of A375 stable cell lines. The first group was A375 cells expressing the empty
lentiviral vector containing control shRNA. The second cell line was A375 cells with lentiviral
vector containing UVRAG-specific shRNA, which served as UVRAG knockdown group. UV
exposure was induced at the indicated dose to both of the two groups, followed by colony
formation assay (Figure 5A). Cell survival rate was compared at different doses of UV treatment.
The red color is UVRAG knockdown group and the black color is the control group. Comparing
the two different groups, we found that under different doses of UV treatment, the UVRAG
knockdown group showed a significant reduction of cell survival rate compare to the control
group. To further confirm our finding, we treated the control group cells and UVRAG
knockdown cells with two different UV-mimic drugs, NFZ and 4-nitroquinoline 1-oxide
(4NQO). Both drugs are known to mimic the biological effects of ultraviolet light on various
organisms. We treated the control group cells and UVRAG knockdown group cells with the
drugs. The survival rate of UVRAG-knockdown A375 cell (red color) was drastically decreased
when compared with the control group (black color) (Figure 5B and 5C). However, considering
26
the possibility that UVRAG shRNA may have “off-target” effects, we re-expressed shRNA-
resistant UVRAG in UVRAG knockdown cells, creating a stable cell line serving as the UVRAG
rescued group. As shown in the colony survival assay, we found that re-expression of UVRAG
in UVRAG knockdown group (green color) significantly increased the survival rate of A375 for
UV radiation at different UV dose treatment (Figure 5A). This result indicated that this was a
UVRAG-specific effect. Moreover, we found that the rescue group compared with the control
group, shared the same survival rate (Figure 5A). We also use UV mimic drugs NFZ and 4NQO
to confirm this finding(Figure 5D). Similarly, UVRAG-knockdown cells were successfully
rescued by the re-expression of UVRAG
and the survival rate was similar to control group
(Figure 5B and 5C). These findings, it indicated that UVRAG plays an essential role in the
protection of UV-induced damage.
We also conducted a gain-of-function experiment to further validate our finding. First,
two A375 stable cell line were established. A375 cells stably expressing UVRAG served as the
ectopic expression group and the A375 cells containing an empty vector served as the control
group. Cells were treated with UVR as indicated, followed by colony forming assay. We found
that the UVRAG overexpression group showed a significant survival rate increase when
compared with the control group. This was also consistent with the result of UV mimic drugs
treatment (Figure 5D-5F). Thus, it was further validated that UVRAG is able to protect cells
from UV damage.
Although we have shown that UVRAG can protect cells from UV-induced damage, we
could not rule out the possibility that UVRAG also participates in protecting against other types
of damage. Thus we Methyl methanesulfonate (MMS), a DNA replication inhibitor and
Camptothecin (CPT), a DNA topoisomerase inhibitor, to test UVRAG protection. We compared
27
the survival rate of UVRAG-knockdown group and control group cells. As shown in Figure 5H
and 5I, after the drug treatment, the survival rate of UVRAG-knockdown group and control
group showed a similar decreasing pattern and there were no significant difference detected. We
also compared the survival rate of UVRAG re-expression group. Similar there were no
significant differences between the control group, UVRAG knockdown group and UVRAG re-
expression group. This suggested that UVRAG is involved in UV damage repair. However, in
other kinds of cell damage, it showed that the effect of UVRAG depletion was not as great in
Figure A-F.
Next we performed a loss-of-function experiment, by comparing the survival rate of
UVRAG knockdown cells and control group cells We found that UVRAG knockdown cells had
a significant reduction in survival rate, proving that UVRAG plays an essential role in protecting
cells from UV-induced damage. Re-expression of UVRAG successfully rescued the UVRAG
knockdown cells excluding the “off-target” effects of shRNA. We performed a gain-of-function
experiment and found that ectopic expression of UVRAG in A375 cells can significantly
increase survival rate compared with control group. To further test this finding, we also used
different drugs to exclude the potential possibility that UVRAG may participate in other damage
protection. In general, we show that UVRAG plays an essential role in UV-induced damage.
28
Figure 5 The Role of UVRAG In UV-irradiation Sensitivity
(A-C, H-J) UV sensitivity of A375 cells upon UVRAG inhibition. Three A375 stable cell line
were established. The first two are A375 stable cell line containing control shRNA and A375
stable cell line expressing UVRAG specific shRNA. The third is established by transferring
retroviral vector containing UVRAG into A375 stable cell line which expressed UVRAG
specific shRNA. Cells were exposed to the indicated doses of UV-C, NFZ and 4-NQO, or CPT
and MMS, followed by colony survival assay. Data are mean ± SD from three independent
experiments. *p < 0.05; **p < 0.01; ****p < 0.0001 (UVRAG shRNA_Vector vs. Control
shRNA). (D-G) Two A375 stable cell line were established that one was transferred with flag-
tagged empty vector and the other one was with WT UVRAG, followed by indicated doses of
UV-C irradiation or NFZ and 4-NQO and colony survival assay. UVRAG expression was
assessed by immunoblotting. Data are mean ± SD from three independent experiments. * p <
0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (UVRAG_WT vs. Vector). These figures
were conducted with Dr. Yongfei Yang.
29
3.2 UVRAG Is Required for UV-induced Photolesion Repair
Previously we found that UVRAG plays a role in the UV radiation resistance mechanism.
Considering the multiple pathways in the UV resistance mechanism, we wanted to find exact
position of UVRAG. As introduced before, chromatin-associated cyclobutane pyrimidine dimer
(CPD) is a sensitive and representative marker of UV-induced DNA damage, which occupies
about 75% of the UV-induced damage in DNA. Thus, we use CPDs as a marker to examine
whether UVRAG is involved in DNA photolesion repair mechanism.
First, we stained the CPDs and nuclei in A375 UVRAG knockdown stable cell line and
A375 control cell line at different time points before and after UV treatment. CPDs are marked
as red and nuclei are marked as blue. As shown in the first row of Figure 6A,, there were no
CPDs in both control group and UVRAG knockdown group before the UV treatment. After the
UV treatment and 5 minutes recovery of cells, we stained the cells with CPD specific antibody.
As shown in the second row in Figure 6A, both control group and UVRAG knockdown group
had similar amount of CPDs staining. Next, we compared the CPDs after a 6-hours recovery. As
shown in the third row of Figure 6A, CPDs in control group had a dramatic decrease at 6 hrs
when compared with 5 min stage. CPDs in UVRAG knockdown group almost remained the
same when compared with 5 min stage. As shown in Figure 6B, CPDs of the UVRAG
knockdown group at 6 hours still remained high when compared with the control group, which
has already decreased to around 80% (Figure 6B). Thus, the control group has a significant
decrease of CPDs at 6 hr compared with UVRAG knockdown group. When we compared CPDs
at 24 hrs, the control group decreased when compared with CPDs at 5min and 6hrs. However, as
shown in the fourth row of Figure 6A, CPDs in UVRAG knockdown group still remained high
when compared at 5 min and 6 hrs. We found that CPDs in the control group had already
30
decreased to around 40% compared with around 90% in UVRAG knockdown group (Figure 6B).
Thus, CPDs in the control group showed a tendency to decrease and CPDs in the UVRAG
knockdown group remained high at the time points of 6 hrs, 12 hrs and 24 hrs. The decreasing of
CPDs in control group was also significant when compared with that in UVRAG knockdown
group (Figure 6B). In order to further support the finding, we measured CPDs levels in UVRAG
rescue group stable cell lines. Similarly, UVRAG rescue group showed the same amount of
CPDs at 5 min compared with control group and UVRAG knockdown group (Figure 6A).
However, as shown in the third row of Figure 6A, when we compared the UVRAG knockdown
group with UVRAG rescue group, we found that CPDs in UVRAG rescue group had a
significant decrease compared with CPDs in UVRAG knockdown group at 6 hr time point,
which is consistent with that in the control (Figure 6B). At the time point of 24 hrs, as shown in
the fourth row of Figure 6A, CPDs in UVRAG rescue group was consistently decreasing.
Similarly, the percentage of remaining CPDs in control group and rescue group were all near
40% compared to 90% in UVRAG knockdown group at 24 hrs (Figure 6B). Thus, combining all
these result, we showed that UVRAG is required in DNA photolesion repair.
In order to further test these findings, we performed the same experiments in UVRAG
ectopic expression cells and control cells. As shown in the second row of Figure 6D, at the time
point of 5 min, UVRAG ectopic expression group and control group showed the same
percentage of CPDs. However, at the time point of 6 hr recovery, ectopic expression of UVRAG
enhanced the clearance rate of CPDs when compared with control group (Figure 6D). As shown
in Figure 6E, the remaining CPDs in the control group was near 80% and that in ectopic
expression group was near 60%. Similarly, the CPDs have almost gone in the ectopic expression
group compared with that in control group at the 24 hrs (Figure 6D). The percentage of
31
remaining CPDs were 20% in ectopic expression group compared with 50% in control group at
24 hrs (Figure 6E).
Thus, we used loss-of-function and gain-of-function experiments to show that UVRAG is
involved in DNA photolesion repair. By comparing the CPDs in UVRAG knockdown group and
control group, we found that UVRAG has a significant effect on the clearance rate of CPDs.
Moreover, when rescued, UVRAG knockdown group can recovery the same clearance rate as
control group. By comparing the control group with UVRAG ectopic expression group, we also
found that overexpression of UVRAG can promote clearance of CPDs. These results support the
theory that UVRAG participates in DNA photolesion repair caused by UV.
32
33
Figure 6 UVRAG Is Required for UV-induced Photolesion Repair
(A-C) Three A375 stable cell lines were used, A375 stable cell line containing control shRNA
and A375 stable cell line expressing UVRAG specific shRNA, as well as the re-expression of
UVRAG in UVRAG knock down A375 cells. (D-E) Two A375 stable cell line were used. One
was transferred with flag-tagged empty vector and the other one was with WT UVRAG. Cells
were treated by UV-C and different period of recovery time was documented as indicated.
Representative images are shown. Quantification of the percentage of remaining CPD per cell
relative to that of 0 hr after UV-C in each sample is plotted. UVRAG expression was assessed by
immunoblotting and compared to actin levels. Data shown represent mean ± SD; n = 200 cells,
data pooled from three independent experiments. Scale bar, 20 µm. *p < 0.05; ****p < 0.0001
(UVRAG shRNA_Vector vs. Control shRNA)
3.3 The Involvement of UVRAG In UV-induced Photolesion Repair
is Independent of Autophagy
As previously mentioned in the introduction, although UVRAG was first identified as a
UV related gene, it was also identified as an important regulator in autophagy. Thus, we wanted
to determine if UVRAG is also involved in the photolesion repair through autophagy.
Autophagy-deficient Atg5-knockout immortalized MEFs (iMEF) cells and Atg5 wild-
type marked as Atg5
-/-
and
Atg5
+/+
, respectively were used for this study. Atg5 is critical in the
elongation of the autophagasome and deletion of Atg5 blocks the autophagy (Codogno et al.,
2006). Both of the immortalized MEF cells stably expressing either control shRNA vector or
UVRA-shRNA were treated with indicated UV-irradiation. After the treatment, Atg5
+/+
UVRAG
knockdown cells (red color) and Atg5
-/-
UVRAG knockdown cells (blue color) had a high
remaining of CPDs at the different repair time points (Figure 7A). However, Atg5
+/+
control
group (black color) and Atg5
-/-
UVRAG control group (green color) had a significant high
clearance rate of CPDs at every time point. For example, approximately 100% of CPDs remained
in both of the UVRAG knockdown groups compared with 70% in both of the control groups at 6
hrs (Figure 7A). Similarly, approximately 90% of CPD remained in both of the UVRAG
knockdown groups compared to 40% in both of the control groups at the time point of 24 hrs.
34
Thus, autophagy does not play a role in the clearance of CPDs and the inhibition of autophagy
cannot prevent the clearance of CPDs mediated by UVRAG. On the contrary, the clearance of
CPDs would be affected if UVRAG was knock down, whether or not autophagy was inhibited.
To further test whether UVRAG participates in UV-induced DNA damage repair
independent of its autophagic function, we used Bafilomycin A1 (Baf-A1) to treat A375 control
cells and UVRAG knockdown cells. Baf-A1 is used to block autophagosome degradation and
inhibiting autophagy (Yamamoto et al., 1998). We compared the CPD levels at different time
points after treating with the indicated dose. As shown in the third row of Figure 7D, the control
group treated with DMSO or Baf-A1 both showed a significant reduction of CPDs at the time
point of 6 hr compared with that in 5 min. However, UVRAG knockdown group treated with
DMSO or Baf-A1 showed no significant difference in CPD level between 6 hrs and 5 min.
Similarly, the control group treated with DMSO or Baf-A1 showed the same tendency of CPDs
clearance and UVRAG knockdown group treated with DMSO or Baf-A1 showed high levels of
remaining CPDs at 24 hrs. The CPD staining in control group cells was around 40% but
UVRAG knockdown cells remained around 80% of CPD at 24 hr, (Figure 7C).
Taken together, we found that UVRAG is directly involved in the photolesion repair
independent of autophagy.
35
Figure 7 UVRAG is Involved In UV-induced Photolesion Repair is Independent of
Autophagy
36
(A and B) Control shRNA or UVRAG-specific shRNA were transferred into the Atg5+/+ and
Atg5-/- immortalized MEF cells and then subjected to UV-C. (C and D) A375 stable cell line
contains control shRNA and A375 stable cell line expressed UVRAG specific shRNA were
treated with indicated dose of DMSO and Baf-A1. The percent distribution of CPD foci before
UV and drug treatment, and 5 min, 6 hrs, and 24 hrs post-UV was determined. Protein levels of
UVRAG and Atg5 are shown.Scale bar, 20 µm. Data are mean ± SD from three independent
experiments. ****p < 0.0001 (Atg5-/- UVRAG shRNA vs. Atg5-/- Control shRNA), (Baf-A1
UVRAG shRNA vs. Baf-A1 Control shRNA).
3.4 UVRAG Regulates GG-NER Pathway
UV induced DNA damage is mainly repaired by NER pathway. There are two sub-
pathways, GG-NER and TC-NER. The main difference between these two sub-pathways is the
recognition step, whereby GG-NER sub-pathway recognizes both the transcription and non-
transcription region, while TC-NER pathway only recognizes damaged DNA in the transcription
region. Thus, we wanted to determine the role of UVRAG in both pathways.
In order to test the role of UVRAG in the two different sub-pathways, we used
unscheduled DNA synthesis (UDS) and recovery of RNA synthesis (RRS) assays (Kelly &
Latimer. 2005; Yamada et al., 2002). UDS has been well established as a method to detect GG-
NER activity and RRS has been established as a method to detect the TC-NER. We included,
GG-NER deficiency cells lacking critical GG-NER recognition component DDB2 and XPC,
which are TC-NER deficiency cells lacking critical TC-NER component CSA, and NER-
deficient cells lacking XPA to served as control groups.
UDS uses 5-ethynyl-2’-deoxyuridine (EdU) as a marker for GG-NER pathway activity
because EdU can participate in DNA synthesis in the GG-NER repair pathway. However, Edu
can also participate in DNA replication. The difference between EdU in GG-NER and DNA
replication is that EdU in DNA replication has a brighter fluorescence than that in GG-NER
pathway. As shown in Figure 8A, the circled cells indicate EdU participation in DNA replication
37
and were not counted as the GG-NER repair cells. The cells marked with a star represent the
activation of GG-NER and were quantified.
Two established stable cell lines, A375 UVRAG knockdown stable cell line and the
A375 control cell line were used to examine the role of UVRAG in GG-NER pathway. As
shown in the first row of Figure 8A, before the UV treatment, EdU only participated in the DNA
replication and there is no GG-NER repair in both groups. After UV treatment, as shown in the
second row of Figure 8A, both of the groups showed that GG-NER pathway activation. However,
when we compared the control group and UVRAG knockdown group, we found that the
activated GG-NER repair (presented by the star) in UVRAG knockdown group is much less than
that in control group.
Furthermore, we compared the fluorescence intensity and found that fluorescence
intensity of UVRAG knockdown group was reduced compared with control group. In order to
compare fluorescence intensity after the UV treatment, we compared four different A375 cell
lines: A375 UVRAG knockdown stable cell line, A375 shRNA control cell line, A375 UVRAG
ectopic expression cell line and A375 UVARG expression control cell line. As shown in Figure
8B, after the UV treatment, all four groups had increased fluorescence intensity (filled bar)
compared with cells that without UV treatment (blank bar). We first compared UVRAG ectopic
expression group and its control group. After the UV treatment, the ectopic expression of
UVRAG showed a significant higher rate of fluorescence intensity than the control group (Figure
8B). Then we compared the UVRAG knockdown group and its control group. We found that
UVRAG knockdown caused a significant decrease in the UDS when compared with its control
group (Figure 8B). Thus, combining the result from four different cell lines, UVRAG plays a
role in GG-NER pathway.
38
Next, we used the RRS assay to examine whether or not UVRAG is involved in TC-NER
pathway. RRS uses 5-ethynyl uridine (EU) as a marker for TC-NER pathway. After the UV
treatment, cells had a 4-hour recovery. Then we used EU to detect the transcription activities.
The fluorescence represents the transcription activity. As introduced before, RNA polymerase
detects the DNA lesion and RNA polymerase activity is inhibited at the DNA lesion. Therefore,
if TC-NER pathway is damaged, which in turn, transcription activity is inhibited, resulting in a
reduction of fluorescence intensity. First, we compared A375 UVRAG knockdown stable cell
line and the A375 control cell line. As shown in the first row of Figure 8D, both groups have
similar transcription without UV treatment. After UV treatment, we found that UVRAG
knockdown group did not lead to a significant reduction in the TC-NER activity when we
compared the fluoresce intensity in control group and UVRAG knockdown group. Similarly, as
shown in Figure 8C, ectopic expression of UVRAG did not cause a significant increase in
fluorescence intensity compared with the control group, which means TC-NER activity was not
increased by overexpressed UVRAG. Contrary to the USD, RRS showed that UVRAG had
much less of an effect in the TC-NER pathway.
We found that in UDS method, UVRAG knockdown influenced GG-NER pathway
activity. When UVRAG was ectopically expressed, GG-NER activity was significantly increased
compared with its control group. However, RRS showed that no significant difference in TC-
NER between the UVRAG knockdown group and control group. Similarly, ectopic expression of
UVRAG did not increase TC-NER repair. RRS cannot rule out the role of UVRAG in TC-NER
pathway, but can show that UVRAG plays an important role in GG-NER pathway.
39
Figure 8 UVRAG Regulates GG-NER Pathway
(A to D) A375 UVRAG knockdown stable cell line, the A375 shRNA control cell line, UVRAG
ectopic expression group and UVARG expression control group are treated by UV-C-irradiated
40
and subjected to UDS and RRS assays. Control groups have been divided into HDFα cells,
DDB2-deficient, XPC-deficient, CSA-deficient, and XPA-deficient cells. Filled bars, UV-
irradiated; open bars, no UV.. Data shown represent mean ± SD from three independent
experiments. *p < 0.05; **p < 0.01; n.s., not significant.
41
Discussion
In the present study, I have demonstrated that UVRAG, a multi-function protein, plays an
important role in protecting cells from UV damage. Furthermore, I found that this protection is
due to UVRAG involvement in DNA repair GG-NER pathway and that this process is
independent of autophagy.
Previously, the functional exploration of UVRAG was mainly related to autophagy and
tumor suppression. For example, UVRAG is involved in the promotion of autophagosome
formation when interacting with Beclin1. In this study, I explored the relationship between
UVRAG and its role in UV damage protection. It has been shown that UVRAG plays an
important role in the UV resistance mechanism. In a loss-of-function experiment, when UVRAG
was knock down, UV treated cells had lower survival rate compared with control group (Figure
5A). Meanwhile, cells overexpressing UVRAG showed an increase in the survival rate compared
with its control cells (Figure 5C). Thus, UVRAG is involved in cell protection induced by UV
radiation.
Next we explored whether UVRAG participated in the UV-induced DNA damage repair.
CPDs are considered one of the most critical characteristics of UV damaged DNA. In this study,
CPD distribution was analyzed in UVRAG-knockdown cells. CPD remaining in UVRAG-
knockdown cells was high compared with control group after UV treatment. The UVRAG
knockdown cells had high remaining levels of CPDs compared with control group at different
time points, 6h, 12 hrs and 24hrs, (Figure 6A, 6B). Similarly, in ectopic expression of UVRAG
group, the CPD clearance rate was increased compared with control cells (Figure 6D). UVRAG-
knockdown cells can be rescued so that the clearance rate of CPDs had a significant increased
compared with UVRAG knockdown group (Figure 6A). Moreover, the clearance by rescue is the
42
same as the control group. This proves that UVRAG is involved in the UV-induced DNA repair
mechanism.
There are two different recognition models of NER pathway, GG-NER pathway and TC-
NER pathway. By using UDS and RRS assays, we found that UVRAG is involved in the GG-
NER pathway (Figure 8A and 8B). Compared with control group cells, the fluorescence intensity
in UVRAG knockdown group had a significant reduction. This means that UVRAG knockdown
had an important negative effect in GG-NER function. Ectopic expression of UVRAG increased
the fluorescence intensity, indicating that ectopic expression of UVRAG can increase GG-NER
activity (Figure 8A and 8B). Thus, this supports that UVRAG participation in the UV-induced
DNA damaged repair by GG-NER pathway. We used RRS assay to test UVRAG in TC-NER
function. After the UV treatment, transcription activities in UVRAG knockdown cells and
control cells showed no significant difference (Figure 8C and 8D). UVRAG knockdown does not
have a significant effect on TC-NER repair because the transcription activity is related to TC-
NER repair ability after UV treatment. Similarly, ectopic expression of UVRAG did not affect
transcription activity, which means that ectopic expression of UVRAG did not affect the TC-
NER pathway. This data shows that UVRAG plays an important role in GG-NER pathway
although it cannot exclude the role of UVRAG in the TC-NER pathway.
We wanted to determine whether autophagy is involved in the DNA damage repair
because UVRAG is an important regulator of autophagy. We found that in Atg5
+/+
and Atg5
-/-
UVRAG knockdown cells, CPD remained high at different repair time points (Figure 7A). In
Atg5
+/+
and Atg5
-/-
control group cells, there was significantly high CPD clearance compared
with UVRAG knockdown iMEF cells. This was consistent with the autophagy inhibitor
treatment results (Figure 7C and 7D). In conclusion, UVRAG directly joins the photolesion
43
repair mechanism independent of autophagy, although this cannot rule out the role of autophagy
in UV-resistance mechanism.
44
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Abstract (if available)
Abstract
DNA damage caused by ultraviolet radiation (UVR) is the leading factor in skin cancer development. Nucleotide excision repair (NER) is one of the main mechanisms responsible for repairing UV-induced DNA damage caused by UV. UV-radiation Resistance Associated Gene (UVRAG), though first identified as a UV-related gene, is generally known as an important regulator in different cellular pathways such as autophagy and intracellular membrane trafficking. Here, I identify the essential role of UVRAG in UV-induced photolesion repair. This is mainly through the regulation of the global-genomic nucleotide excision repair pathway (GG-NER). And this regulation is independent of autophagy. These findings demonstrate that UVRAG is essential in UV-resistance through participation in the GG-NER pathway.
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Wang, Qiaoxiu
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Core Title
UVRAG protects cells from UV-induced DNA damage by regulating global genomic nucleotide excision repair pathway
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Keck School of Medicine
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Master of Science
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Biochemistry and Molecular Biology
Publication Date
07/22/2016
Defense Date
06/10/2016
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DNA damage,NER pathway,OAI-PMH Harvest,UV,UVRAG
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Tokes, Zoltan A. (
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DNA damage
NER pathway
UV
UVRAG