University of Southern California Dissertations and Theses

Mechanism of human nonhomologous DNA end joining

(USC Thesis Other)

Mechanism of human nonhomologous DNA end joining

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

MECHANISM OF HUMAN NONHOMOLOGOUS DNA END JOINING

by

Jiafeng Gu

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

August 2009

Copyright 2009 Jiafeng Gu

ii
ACKNOWLEDGEMENTS

First, I would like to thank my advisor Dr. Michael Lieber. He works harder
than anyone of us in the lab. Whenever I have a problem in technique, an idea
on the experimental design, a random question on the project, he is here to offer
advice and help. I also want to thank him for his support and advice on other
topics besides my graduate studies.
In addition, I would like to acknowledge Dr. Chih-Lin Hsieh for her
generous guidance and support during my graduate studies. Her incredible
knowledge is a wealth for the lab. I learned a lot in the lab meeting when she was
present.
I would also like to thank my committee members for their guidance and
support throughout my graduate studies: Dr. Steven Goodman, Dr. Xiaojiang
Chen, Dr. Oscar Aparicio, and Dr. Ebrahim Zandi.
I would also like to take this opportunity to thank former and present
members from Lieber lab: Dr. Sathees Raghavan, Dr. Yunmei Ma, Dr. Kefei Yu,
Dr. Haihui Lu, Dr. Noriko Shimazaki, Dr. Feng-ting Huang, Dr. Xiaoping Cui, Dr.
Albert Tsai, Dr. Deepankar Roy, Go Watanabe, Sicong Li, Zheng Zhang, and
Zhengfei Lu.
I also owe my deepest gratitude to my family, my parents, Qirong Gu and
Guoying Zhang, and my little brother, Jiaxiang Gu. Their love, support and
understanding give me the courage to pursue my dream here.

iii
Last but not least, I would like to express my thanks to the most important
person in my life, my wife, Lin Xu. Her love, support and understanding helped
me go through the tough times I had over the years. I can never thank her
enough.

iv
TABLE OF CONTENTS

Acknowledgements ii
List of Figures vi
Abstract viii
Chapter 1 GENERAL INTRODUCTION 1
1. Genomic Instability, DNA Double-strand Break & Repair,
HR and NHEJ 1
2. Genetic perspective of NHEJ pathway in DSB repair 6
3. Biochemical perspective of NHEJ pathway in DSB repair 8
4. Biochemical reconstitution of the NHEJ pathway 14
Chapter 2 EXPERIMENTAL DESIGN AND PREPARATION 16
1. Oligonucleotides 16
2. Protein expression and purification 20
3. Expression and purification of native DNA polymerase mu 21
4. Template independent polymerization on substrates
in free solution 22
5. Template independent polymerization on immobilized
DNA substrates 23
6. DNA ligation assay 24
7. Sequencing of junctions of the ligation products 25
8. Template dependent primer extension assay 26
Chapter 3 TEMPLATE DEPENDENT AND TEMPLATE
INDEPENDENT ADDITION BY POLYMERASE MU 27
Abstract 27
Introduction 27
Results 30
Discussion 54

Chapter 4 FLEXIBLE LIGATION CAPABILITY BY
XRCC4:DNA LIGASE IV 59
Abstract 59
Introduction 60
Results 62
Discussion 82

v
Chapter 5 BIOCHEMICAL FUNCTION OF XLF (CERNUNNOS)
IN NHEJ 91
Abstract 91
Introduction 91
Results 93
Discussion 110
Chapter 6 NHEJ BIOCHEMICAL RECONSTITUTION
AND ANTI-CANCER TREATMENT 113
Bibliography 116

vi
LIST OF FIGURES

Figure 1.1 Schematic diagrams of major NHEJ components. 4
Figure 3.1 Template independent polymerase activity of
polymerase mu on substrates in free solution. 33
Figure 3.2 Template independent polymerase activity of
polymerase mu provides short terminal
microhomology for ligation. 35
Figure 3.3 Template dependent primer extension activity
of polymerase mu and polymerase lambda. 40
Figure 3.4 Template independent synthesis by polymerase
mu on immobilized DNA substrates distributed
at low density on agarose beads. 41
Figure 3.5 Template independent polymerase activity of
polymerase mu on immobilized DNA substrates. 42
Figure 3.6 Ribonucleotide addition by polymerase mu on
immobilized DNA substrates. 43
Figure 3.7 Polymerase mu template independent polymerase
activity provides terminal microhomology for ligation
by XRCC4:DNA ligase IV. 48
Figure 3.8 One base pair of terminal microhomology is
sufficient for direct ligation by XRCC4:DNA ligase IV. 51
Figure 3.9 Phosphate repulsion can decrease the ligation efficiency. 52
Figure 4.1 XRCC4:DNA ligase IV can ligate over a gap. 64
Figure 4.2 XRCC4:DNA ligase IV and Ku can ligate over a gap. 66
Figure 4.3 Time course of ligation of a nick, a 1 nt-gap, and a
fully incompatible DNA end substrate by
XRCC4:DNA ligase IV and Ku. 68

vii
Figure 4.4 XRCC4:DNA ligase IV and Ku can ligate fully
incompatible DNA ends (1). 71
Figure 4.5 XRCC4:DNA ligase IV and Ku can ligate fully
incompatible DNA ends (2). 73
Figure 4.6 Ligase activity comparison with DNA
double-strand break substrates. 77
Figure 4.7 Single-stranded DNA can be ligated by
XRCC4:DNA ligase IV. 80
Figure 4.8 Long dT overhangs can be directly ligated
by XRCC4:DNA ligase IV. 81
Figure 4.9 Function of XRCC4:DNA ligase IV in ligating
incompatible DNA ends. 85
Figure 5.1 DNA substrates with a gap are ligated by
XRCC4:DNA ligase IV in a sequence
dependent manner. 96
Figure 5.2 Sequence dependence of joining by
XRCC4:DNA ligase IV. 101
Figure 5.3 Steric hindrance model for terminal sequence effects
on ligation efficiency by XRCC4:DNA ligase IV. 103
Figure 5.4 XLF stimulates incompatible end ligation by
XRCC4:DNA ligase IV but not compatible and
blunt end ligations. 106
Figure 5.5 XLF stimulation of ligation as a function of
Mg
2+
concentration. 108
Figure 5.6 XLF increases the ligation efficiency for incompatible
DNA ends joined by XRCC4:DNA ligase IV. 109

viii
ABSTRACT
DNA double-strand breaks (DSBs) represent the most deleterious form of
DNA damage, as both of the DNA strands are broken. In mammalian cells, DSBs
are repaired predominantly by nonhomologous DNA end joining (NHEJ)
pathway. NHEJ functions throughout the cell cycle to repair such lesions. Defects
in NHEJ result in marked sensitivity to ionizing radiation and ablation of
lymphocytes, which rely on NHEJ to complete the rearrangement of antigen-
receptor genes. NHEJ is typically imprecise, a characteristic that is useful for
immune diversification in lymphocytes, but which might also contribute to some
of the genetic changes that underlie cancer and aging. To further understand the
mechanism of human nonhomologous DNA end joining pathway, we performed
in vitro biochemical reconstitution assay. Here, we present some distinctive
features of polymerase mu and the ligase complex, XRCC4:DNA ligase IV:XLF.
Polymerase mu has both template dependent and template independent
addition capabilities on DSB end substrate under physiological conditions.
Template independent addition by polymerase mu prefers to add pyrimidines
onto the DNA ends. In addition, template independent addition onto the 3’
overhang of the DNA substrates by polymerase mu provides terminal
microhomology for ligation by Ku and XRCC4:DNA ligase IV.
XRCC4 and DNA ligase IV form a complex that is essential for the repair
of all double-strand DNA breaks by the NHEJ pathway in eukaryotes. We find
here that, in the absence of processing factors, XRCC4:DNA ligase IV can ligate

ix
two double-strand DNA ends that have fully incompatible short 3’ overhang
configurations with no potential for base pairing. Moreover, at DNA ends that
share 1–4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of
1 nucleotide. XLF stimulates the joining of both incompatible DNA ends and
compatible DNA ends at physiological concentrations of Mg
2+
. Hence, the
remarkable flexibility of the ligase complex is paramount in nonhomologous DNA
end joining. These observations provide an explanation for several in vivo
observations that were difficult to understand previously. Furthermore, those
unusual ligation capabilities of XRCC4:DNA ligase IV:XLF complex may provide
useful applications in recombinant DNA technology.

1
CHAPTER 1
GENERAL INTRODUCTION

1. Genomic Instability, DNA Double-strand Break & Repair, HR and NHEJ
DNA double-strand breaks (DSBs) represent the most deleterious form of
DNA damage, as both of the DNA strands are broken. Even one single unrepaired
DSB can lead to cell death (Rich et al., 2000), while misrepaired DSBs can lead to
chromosomal translocations and other genomic rearrangements which underlie
cancer and aging (Hoeijmakers, 2001; van Heemst et al., 2007). All those permit
the contribution of DSB repair in the maintenance of genomic integrity.
Both pathological and physiological events in the cells can cause DNA
DSBs. Pathologically, DNA DSBs can be generated by ionizing radiation (IR),
chemotherapeutic drugs such as topoisomerase inhibitors, and stalled replication
forks; physiologically, DNA DSBs can be introduced during normal cellular
processes like V(D)J recombination and class switch recombination (Lieber et al.,
2003).
Two mechanistically distinctive repair pathways have evolved to mend
these breaks in eukaryotes: homologous recombination (HR) and
nonhomologous DNA end-joining (NHEJ). HR is widely regarded as an accurate
method to repair DSBs, which requires an intact sister chromatid as the DNA

2
template. Due to this special requirement, HR is restricted to late S or G2 of the
cell cycle when a sister chromatid is available after DNA replication. In contrast,
NHEJ simply rejoins the broken DNA ends directly, and functions throughout the
cell cycle. However, DSBs, especially those generated by IR, usually contain
different overhang configurations (blunt end, 3’ overhang or 5’ overhang) on the
broken DNA ends, and also may have DNA lesions like 3’-phosphate or
3’-phosphoglycolate groups on the DNA termini. During NHEJ, those overhang
configurations are processed by nucleases or polymerases before ligation which
results in nucleotide loss or addition in the junctions. NHEJ is thus considered as
potentially error-prone: it only restores the physical integrity of the chromosome,
but not the information content. Namely, local DNA information is sacrificed for
genomic integrity. In mammalian cells, DSBs are repaired predominantly by NHEJ
pathway (Lieber, 2008).
The main proteins involved in mammalian NHEJ are the Ku heterodimer
(Ku 70/80 complex), DNA-PKcs (DNA-dependent protein kinase catalytic subunit),
Artemis, XRCC4 (X-ray-complementing Chinese hamster gene 4), DNA ligase IV,
XLF/Cernunnos (XRCC4-like factor), and polymerases lambda and mu.
Schematic diagrams of individual components are presented in Figure 1.1.
After DSB, the choice between HR and NHEJ is still an area of active
investigation. Currently, three factors have been suggested to regulate the usage

3
of DSB repair pathways between HR and NHEJ (Rothkamm et al., 2003; Sonoda
et al., 2006): a, cell cycle stage; b, the nature of DSBs; c, cell type. Further
understanding of the control of HR and NHEJ would provide useful insights into
genetic technologies like gene targeting and may also help develop effective
chemotherapeutic treatment.

4
Figure 1.1 Schematic diagrams of major NHEJ components.
Ku: vWA, von Willebrand domain; Ku core, central DNA-binding core; NLS,
nuclear localization sequences; SAP, SAF-A/B, Acinus and PIAS domain, a
putative chromatin/DNA-binding domain.
DNA-PKcs: JK cluster, N cluster, PQR and ABCDE et al. are the
DNA-PKcs autophosphorylation sites; LRR, leucine rich region; FAT, FRAP
(FKBP12-rapamycin-associated protein), ATM (ataxia-telangiectasia mutated),
TRRAP (transactivation/transformation-domain-associated protein) domain; PI3K,
phosphatidylinositol 3-kinase domain.
XRCC4: 334 - 336 aa, due to alternative splicing.
Ligase IV: K273, active site; AdD, adenylation domain; OBD, OB-fold
domain; BRCT, BRCA1 (breast-cancer susceptibility gene 1) C-terminal domain.
TdT: terminal deoxynucleotidyltransferase.
Diagrams are not drawn to scale.

5
Figure 1.1, continued

6
2. Genetic perspective of NHEJ pathway in DSB repair
On the cellular level, NHEJ proceeds through three intertwined stages: 1)
detection of DSBs and cell signaling for repair (cell cycle arrest); 2) processing of
DSB ends and minimizing information loss; 3) ligation of broken DNA ends and
maintenance of genomic integrity (Mahaney et al., 2009). During this process, cell
cycle arrest is important in providing time for repair and avoiding passing wrong
information to the next generation.
NHEJ not only functions in repair of general DSBs, but also in resolution of
those special breaks generated during V(D)J recombination. Deletion or
inactivation of any of the core NHEJ components (Ku, DNA-PKcs, Artemis,
XRCC4, DNA ligase IV and XLF) induces marked sensitivity to IR and other
DSB-inducing agents, as well as defects in V(D)J recombination.
XRCC4 is necessary for DNA ligase IV stability in vivo, and they form a
tight complex in the cells (Grawunder et al., 1997). This complex represents the
feature complex for NHEJ pathway, since it does not function in any other DNA
repair pathways. Mice deficient in XRCC4 or DNA ligase IV have high degree of
neuronal cell death in the developing nervous system, which may partially result
in early embryonic lethality (Ferguson and Alt, 2001). No patients have been
found with homozygous deletion of XRCC4 or DNA ligase IV genes, which also
indicates they are essential for the viability. Patients with inherited hypomorphic

7
mutations in DNA ligase IV gene exhibit radiosensitive, developmental delay and
immunodeficiency (O'Driscoll et al., 2001; Riballo et al., 1999).
Deficiencies in Ku70 or Ku80 do not cause embryonic lethality in mice but
share other similar phenotypes with XRCC4 or DNA ligase IV deficiencies,
including defects in growth, V(D)J recombination, neurogenesis and premature
cellular senescence (Ferguson and Alt, 2001).
Cells that lack DNA-PKcs are highly radiosensitive and have defects in
V(D)J recombination, specifically in processing of coding joints (Meek et al., 2008).
Inactivation of Artemis in humans results in RS-SCID (radiation sensitive - severe
combined immune deficiency) (Moshous et al., 2001). In lymphocytes, similar to
cells lacking DNA-PKcs, Artemis null cells accumulate unopened coding joint DNA
hairpins during V(D)J recombination. However, although inactivation of Artemis
results in radiosensitive in cells, Artemis nuclease activity is only responsible for
the repair of about 10% of the ionizing radiation induced DSBs (Riballo et al.,
2004).
Polymerases mu and polymerase lambda have been implicated in the
NHEJ pathway through both genetic and biochemical studies. Studies from
polymerase mu knockout mice indicate that polymerase mu function is restricted
in light chain gene rearrangement during immunoglobulin V(D)J recombination in
vivo (Bertocci et al., 2003). Recent studies from the same lab further dissected the

8
roles of polymerase mu, lambda and TdT (terminal
deoxyribonucleotidyltransferase) in V(D)J recombination with single and double
knockout mice (Bertocci et al., 2006). In contrast to polymerase mu, polymerase
lambda was shown to be involved in heavy chain gene rearrangement only,
preceding the function of TdT on the junctions. Those nonoverlapping functions of
polymerase X family during in vivo immunoglobulin V(D)J recombination may
reflect the spatial and temporal expression differences and various recruitment
mechanisms. Interestingly, neither polymerase lambda nor polymerase
lambda-mu double negative cells show increased sensitivity to IR or radiomimetic
drug bleomycin, which indicates that those two polymerases are not essential for
the NHEJ pathway but may be required for repair of a subset of DSBs (Bertocci et
al., 2006).

3. Biochemical perspective of NHEJ pathway in DSB repair
On the biochemical level, NHEJ of DSBs is thought to begin with the
binding of the heterodimer Ku70/80 to the double-stranded DNA (dsDNA) ends.
Two characteristic features of the Ku70/80 complex contribute to this first step in
NHEJ: a), abundance of the Ku70/80 complex in the cell nucleus; b), Ku has high
affinity to bind to the ends of double-stranded DNA. Ku70 and Ku80 central
DNA-binding core form a loop which is suitable for a double-stranded DNA

9
(Walker et al., 2001). After binding to DNA ends, Ku translocates internally away
from the DNA ends, making room for the other processing enzymes, such as
DNA-PKcs (Yoo and Dynan, 1999). Ku-DNA complex and DNA-PKcs form the
DNA-PK complex. Ku and DNA-PKcs only interact in the presence of DNA (Suwa
et al., 1994).
Like many repair processes, NHEJ utilizes nucleases, polymerases, and
ligases; and Ku also functions as a ‘toolbelt’ protein by recruiting each of these
enzymatic components onto the DNA ends (Ma et al., 2004).
The DNA-PK complex may contribute to tethering of the broken DNA ends
together which also protects the DNA termini from unnecessary nuclease attacks.
In addition, protein kinase activity of DNA-PKcs is required for NHEJ in vivo
(Kienker et al., 2000; Kurimasa et al., 1999). Kinase activity of DNA-PKcs is
necessary to activate the endonuclease activity of Artemis (Ma et al., 2002).
Hairpin opening activity of Artemis is important for V(D)J recombination, while 5’
or 3’ endonucleases are involved in overhang processing of general NHEJ.
However, other than DNA-PKcs itself and Artemis, other physiological substrates
of DNA-PKcs kinase activity are still not well defined.
After sensing the DSBs, the next step in NHEJ is to process the complex
DNA ends into ligatable form. Depending on the nature of the break, DSB ends
can vary from one end to another, comprising thousands of different end

10
configurations. Different processing enzymes may be required to remove the
damaged sites or secondary structures. This process is thought to occur in an
iterative mode (Ma et al., 2005a). Processing those end configurations results in
potential nucleotide loss or addition from either of the broken DNA ends; this
makes NHEJ an error-prone repair pathway. During this process, polymerase
lambda and polymerase mu are required to fill in the gaps, while DNA-PKcs
dependent endonuclease activity of Artemis is required to chew back the different
overhang ends.
Artemis itself possesses 5’-3’ exonuclease activity, and in the presence of
DNA-PKcs and ATP, it acquires 5’ endonuclease to trim 5’ overhangs to a blunt
conformation, 3’ endonuclease to trim long 3’ overhangs to much shorter ones,
typically about 4 nucleotides (nt) in length, and hairpin opening activity to open the
hairpins generated by RAG (Recombination Activating Gene) complex during
V(D)J recombination (Ma et al., 2002; Ma et al., 2005b). Those three seemingly
diverse endonucleolytic activities could be unified into single activity if
Artemis:DNA-PKcs complex recognizes 4 nt single-stranded DNA region from a
single-double strand DNA transition and cuts the 3’ side of this 4 nt region. Both in
vivo and in vitro, Artemis is highly phosphorylated by DNA-PKcs or ATM. It was
thought that C-terminal Artemis phophorylation by DNA-PKcs was the
prerequisite for the activation of endonuclease activity in Artemis (Ma et al., 2002;

11
Niewolik et al., 2006). However, recently it has been suggested that rather than
Artemis phosphorylation by DNA-PKcs, autophosphorylation of DNA-PKcs
facilitates the activation of Artemis endonuclease activity, likely by presenting the
DNA substrate in a suitable structure for Artemis (Goodarzi et al., 2006). On the
other hand, the physiological role of Artemis phosphorylation by DNA-PKcs or
ATM is not clear at this moment.
DNA polymerases are thought to be needed for any NHEJ events that
require fill-in of gaps or extension of the 3’ end at 5’ overhangs (Ma et al., 2005a).
The only Polymerase X family member in S. cerevisiae is POL4, which has been
linked to NHEJ repair both genetically and biochemcially (Tseng and Tomkinson,
2002; Wilson and Lieber, 1999). POL4 is most homologous to polymerase mu and
polymerase lambda in mammalian cells (Tseng and Tomkinson, 2002). Three
members of polymerase X family have been suggested to function in NHEJ
pathway, namely polymerase lambda, polymerase mu and TdT. The other Pol X
family member is polymerase beta, which lacks a BRCT [BRCA1 (breast-cancer
susceptibility gene 1) C-terminal] domain and functions in base excision repair.
TdT is only expressed in lymphocytes, which limits its function only to promote
antigen receptor diversity during V(D)J recombination, while polymerase lambda
and polymerase mu are widely expressed and function in general NHEJ pathway.
Immunoinhibition and immunodepletion studies with human nuclear extracts

12
provide evidence that polymerase lambda play a role in NHEJ in its BRCT
dependent manner (Lee et al., 2004). Physical interaction of polymerase mu with
Ku and XRCC4:DNA ligase IV presents polymerase mu as a candidate
polymerase in NHEJ (Mahajan et al., 2002).
Besides polymerase mu, Ku also recruits TdT and polymerase lambda to
the DSB site through their N-terminal BRCT domains (Ma et al., 2004; Mahajan et
al., 1999). On the other hand, polymerase mu is also able to interact with
XRCC4:DNA ligase IV directly in the absence of Ku or DNA (Mahajan et al.,
2002).
Other DNA modifying enzymes are thought to be involved in the NHEJ
processing step, like PNK (polynucleotide kinase) (Chappell et al., 2002; Koch et
al., 2004). However, detailed genetic and biochemical studies are required to
further confirm this.
After broken DNA ends are processed into a ligatable format, those ends
need to be ligated in order to restore the genomic integrity. In NHEJ, XRCC4:DNA
ligase IV complex is responsible for the final ligation step, and it is considered to
be the feature component for the NHEJ pathway (Grawunder et al., 1997; Schar
et al., 1997; Teo and Jackson, 1997; Tomkinson et al., 2006; Wilson et al., 1997).
XRCC4 facilitates DNA end joining by stabilizing DNA ligase IV and stimulating its
activity. XRCC4 interacts with DNA ligase IV through the linker region between

13
two BRCT domains on the C-terminal of ligase IV, which is named “XRCC4
interacting region” (XIR) (Grawunder et al., 1998). Recent crystal structure studies
suggest that XIR is necessary for the interaction between XRCC4 and DNA ligase
IV, but additional residues from the C-terminal distal BRCT domain are required to
stabilize the interaction (Wu et al., 2009). Ku improves the binding of the
XRCC4:DNA ligase IV complex to DNA ends (Chen et al., 2000; Nick McElhinny
et al., 2000).
The recently identified NHEJ factor XLF or Cernunnos forms a
tri-component complex with XRCC4:DNA ligase IV and has been proposed to be
the third essential component in the ligase complex for NHEJ pathway (Ahnesorg
et al., 2006; Buck et al., 2006; Callebaut et al., 2006).
There is no clear boundary between all those three biochemical steps of
NHEJ: sensing, processing and ligation. In fact, in the cells, the NHEJ pathway is
thought to be very flexible (Gu and Lieber, 2008; Lieber et al., 2008). Depends on
the causes of the break, the types of DNA ends and other factors, dynamic
recruitment and release of the various components may occur in a very flexible
manner.

14
4. Biochemical reconstitution of the NHEJ pathway
Researchers have put incredible efforts into the biochemical reconstitutions
of many important cellular pathways. Simply put, there are three stages involved
in this process: first, identification of the individual components of the pathway
through genetic studies; second, purification of each of those components with
gene cloning and protein expression and purification methods; third, biochemical
reconstitution of the pathway using pure forms of the individual components under
physiological conditions. Biochemical reconstitution could help us understand the
molecular mechanism of each component in the pathway. In addition, it could also
provide us the platform to find the small molecule inhibitors of the pathway, which
potentially have therapeutic effects to treat diseases associated with the cellular
pathway.
Over the last decade, there have been many trials to reconstitute the NHEJ
pathway, at least partially. In 2004, our lab developed an elegant and
sophisticated NHEJ reconstitution system, which was the first time to reconstitute
the NHEJ pathway in vitro with all the purified components known at that time (Ma
et al., 2004). This study established polymerase mu and polymerase lambda are
recruited by Ku to the NHEJ pathway with their BRCT domains. Many of the in
vivo findings have been recapitulated in this reconstitution system, including the
junction diversity.

15
However, additional efforts are warranted in the NHEJ reconstitution study.
First, the final ligation product in the above reconstitution system is detected by
PCR (polymerase chain reaction) assay, which may provide some level of
artifacts and mask other findings. Thus, an efficient NHEJ reconstitution system
with ligation detection via direct gel needs to be developed, which would also
facilitate the large scale screening process. Second, in 2006, XLF or Cernunnos
has been identified as the new essential player in NHEJ pathway. Addition of XLF
or Cernunnos into the reconstitution system is therefore necessary.
On the other hand, we would get the benefits from the NHEJ reconstitution:
first, it would help us to further understand the molecular mechanism of each
component and to understand the puzzles from the genetic studies; second, this
reconstitution system would permit us to screen small molecule inhibitors for
NHEJ pathway. Evidence has accumulated that inactivation or inhibition of the
NHEJ pathway would result in chemosensitization or radiosensitization of the
cancer cells (Pastwa and Malinowski, 2007). Therefore, NHEJ inhibitors could be
combined with chemotherapy or radiotherapy to treat cancer patients. In addition,
those small molecule inhibitors may be used in improving the efficiency of gene
targeting, a genetic technology which is dependent on homologous recombination.

16
CHAPTER 2
EXPERIMENTAL DESIGN AND PREPARATION

1. Oligonucleotides
All of the nonbiotinylated oligonucleotides used in this study were
synthesized either by Operon Biotechnologies, Inc. (Huntsville, AL) or Integrated
DNA Technologies, Inc. (San Diego, CA). Biotinylated oligonucleotides were
synthesized by the Microchemical Core Facility (Norris Cancer Center, USC). We
further purified all of the oligonucleotides using 12% or 15% denaturing
polyacrylamide gel electrophoresis (PAGE), and then determined the
concentration spectrophotometrically. DNA sequencing of joined products
confirmed the lack of errors in the oligonucleotide sequences. Possible
contaminants in the oligonucleotide synthesis reagents or in the starting column
for the oligonucleotide syntheses are, according to the manufacturer (Glen
Research, Ann Arbor, MI) below the level that could account for our observed
joined products.
Oligonucleotides were labeled at the 5’-end with [ γ-
32
P] ATP (3000
Ci/mmol) (PerkinElmer Life Sciences, Boston, MA) and T4 polynucleotide kinase
(New England Biolabs, Beverly, MA) according to the manufacturer’s instructions.
Unincorporated radioisotope was removed by using G-25 Sephadex (Amersham

17
Biosciences, Inc., Piscataway, NJ) spin-column chromatography. For the
nonbiotinylated oligonucleotides, to make the double-stranded DNA substrate,
labeled oligonucleotides were mixed with an equal amount of unlabeled
complementary oligonucleotide in a buffer containing 10 mM Tris-hydrochloride,
pH 8.0, 1 mM EDTA, pH 8.0, and 100 mM NaCl. The mixture was heated at
100°C for 5 min, allowed to cool to room temperature for 3h, and then incubated
at 4°C overnight. For the biotinylated oligonucleotides, it is important to note that
labeled oligonucleotides were mixed with half the molar amount of the
complementary biotinylated oligonucleotide (JG193) in a buffer containing 10 mM
Tris-hydrochloride, pH 8.0, 1 mM EDTA, pH 8.0, and 100 mM NaCl. The mixture
was heated at 100°C for 5 min, allowed to cool to room temperature for 3h, and
incubated at 4°C overnight.
The sequences of the oligonucleotides used in this study are as follows:
JG55: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GAG C-3’
JG56: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CAG C-3’
JG67: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GAG-3’
JG68: 5’- CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG

18
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CAG-3’
JG161: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GAG GGG-3’
JG162: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CCC CC-3’
JG163: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GAC C-3’
JG164: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC G-3’
JG165: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA C-3’
JG166: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CAG G-3’
JG169: 5’-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3’
JG171: 5’-GTT AAG TAT GCA TCT CTG CGA TGC ATG TCA CTC AGA CTA
TGG TCA GCG ATC GGC TCG ACC-3’
JG172: 5’-CGA GCC GAT CGC TGA CCA TAG TCT GAG TGA CAT GCA TCG
CAG AGA TGC ATA CTT AAC A-3’
JG173: 5’-GTT AAC TCG CAT GTA GTG TGC CTA CTT GCT CAA GCT GAC
AGC TGT GAC CAG CGC TCG A-3’

19
JG174: 5’-CGA GCG CTG GTC ACA GCT GTC AGC TTG AGC AAG TAG GCA
CAC TAC ATG CGA GTT AAC AGG-3’
JG185: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GA-3’
JG186: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CA-3’
JG191: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GTC-3’
JG192: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CTC-3’
JG193: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CX-3’ (X=biotin)
JG194: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GAG T-3’
JG195: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GAC G-3’
JG196: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG
ACG ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CAC G-3’
JG197: 5’-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GTC C-3’

20
JG198: 5’-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG
ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CTG G-3’
HL3: 5’-CCT CTG AGG GCG AGC CCG AT-3’
YM42: 5’-AGG CTG TGT TAA GTA TCT GCA TTT TTT CGG ATC GGG CTC
GCC CTC AGA GG-3’
YM68: 5’-GAT CCT TCT GTA GGA CTC AGT-3’
YM130: 5’-TTT TTT TTT TTT TTT ACT GAG TCC TAC AGA AGG AT-3’
YM145: 5’-TTT TTT TTT TTT TTT TTT TT-3’
YM149: 5’-ACT GAG TCC TAC AGA AGG ATC TTT TTT TTT TTT TTT-3’

2. Protein expression and purification
The purification of Ku and DNA-PKcs has been described (Ma et al., 2002).
XRCC4:DNA ligase IV complex was purified from baculovirus-insect cell system
as described (Nick McElhinny et al., 2000). Native DNA polymerase mu was
expressed and purified from E. coli, as described previously and in more details
below (Tippin et al., 2004). We made some modifications to eliminate potential
exonuclease contamination as described in detail below. Polymerase lambda was
purified as a recombinant protein from E. coli as described (Shimazaki et al.,
2002). Soluble human XLF-myc-his protein was expressed in 293T cells and
purified by Ni-NTA agarose beads and Mono Q column as described (Lu et al.,

21
2007). DNA ligase I was a gift of Dr. Alan Tomkinson (U. Maryland, Baltimore,
MD). DNA ligase III-alpha was a gift of Dr. Ulf Grawunder (U. Basel, Switzerland).

3. Expression and purification of native DNA polymerase mu
Native DNA polymerase mu expression and purification were described
previously (Tippin et al., 2004). We added some modifications to eliminate
potential exonuclease contamination. Briefly, polymerase mu cDNA was
subcloned into vector pET41b (Novagen) and the recombinant plasmid was used
to express polymerase mu in strain BL21(DE3)R1L Codon Plus (Stratagene).
Cells were grown at 30°C in LB medium supplemented with kanamycin (5 ug/mL)
and chloramphenicol (30 ug/mL) to OD600 of 0.6. Protein expression was
induced with isopropyl-1-thio- β-D-galactopyranoside (1 mM) for 3 additional hours.
Harvested cells were resuspended in lysis buffer (50 mM Tris-Cl (pH 7.5), 1 M
NaCl, 2 mM DTT, and 10% sucrose) and lysed with lysozyme (2 mg/mL) with
stirring for 1 h at 4°C. Soluble protein was recovered by centrifugation (12,000
rpm 30 min in a SS-34 rotor at 4°C). The remaining steps were all carried out at
4°C. Ammonium sulfate was added to 40% saturation, and the protein was
recovered by centrifugation (12,000 rpm 30 min in a SS-34 rotor at 4°C). We then
resuspended the pellet in PC buffer (50 mM Tris-Cl (pH 7.5), 10% glycerol, 1 mM
EDTA, 2 mM DTT) supplemented with 500 mM NaCl and dialyzed against the

22
same buffer overnight. The dialyzed sample was diluted in PC buffer to 125 mM
NaCl, and immediately loaded onto a Whatman P11 phosphocellulose column.
The column was washed for 20 column volumes with PC buffer supplemented
with 125 mM NaCl, and the protein was eluted with a 125 mM to 500 mM NaCl
gradient over 10 column volumes. Polymerase mu containing fractions were
pooled and concentrated using a 60% ammonium sulfate cut, and then
resuspended and dialyzed in PC buffer supplemented with 250 mM NaCl, and
loaded onto a Sephacryl 200 column (Amersham Biosciences) equilibrated with
the same buffer. The purest peak fractions were then loaded onto a Mono S
column (Amersham Biosciences). The column was washed for 15 column
volumes with PC buffer supplemented with 100 mM NaCl, and the protein was
eluted with a 100 mM to 1 M NaCl gradient over 20 column volumes. The eluted
fractions were aliquoted and stored at -80°C.

4. Template independent polymerization on substrates in free solution
The test for terminal deoxynucleotidyl transferase activity in free-solution
was performed in a 10 uL reaction. 20 nM of the DNA substrates were first
incubated with or without protein Ku in 1X ligation reaction buffer (25 mM
Tris-hydrochloride, pH 7.5, 75 mM NaCl, 72.5 mM KCl, 2 mM DTT, 0.025% Triton
X-100, and 100 uM EDTA) supplemented with 10% PEG (MW>8,000D), 50

23
ug/mL BSA, and 5% glycerol at room temperature for 15 min. Nucleotide addition
was initiated by adding 100 µM of each dNTP, 10 mM MgCl
2
with different
combinations of proteins as indicated. Reactions were then incubated at 37°C for
1 hour. After incubation, reactions were stopped, deproteinized by organic
extraction, and analyzed on 11% denaturing PAGE gel. Gels were dried, exposed
in a PhosphorImager cassette, and the screen was scanned in a Molecular
Dynamics PhosphorImager.

5. Template independent polymerization on immobilized DNA substrates
10 pmol of biotinylated DNA substrates with an annealing ratio of 1:2 as
described above were mixed with 50 ul of streptavidin-agarose suspended in 10
mL of 1X ligation reaction buffer (25 mM Tris-hydrochloride, pH 7.5, 75 mM NaCl,
72.5 mM KCl, 2 mM DTT, 0.025% Triton X-100, and 100 uM EDTA). The mixture
was incubated at room temperature on a rotator for 3 hour. After incubation,
agarose beads were collected by low-speed centrifugation (1,000g 5 min) and
washed more than 4 times with 5 mL of 1X ligation reaction buffer until the
radiolabel level in the supernatant from the centrifugation was close to the
background level. Then 50 uL agarose beads were mixed with 50 uL of 1X ligation
reaction buffer. The polymerization test using bead-bound substrate was
performed in a 20uL reaction. Biotinylated DNA substrates (20 nM) were first

24
incubated with or without protein Ku in 1X ligation reaction buffer supplemented
with 10% PEG (MW>8,000D), 50 ug/mL BSA, and 5% glycerol at room
temperature for 15 min. Nucleotide addition was initiated by adding dNTP at 500
uM or 5 mM and 10 mM MgCl
2
with different combinations of proteins as indicated.
Reactions were then incubated at 37°C for 1 hour. After incubation, reactions
were heated to 100°C for 5 min to denature proteins (and specifically to disrupt
the biotin-streptavidin association), deproteinized with organic extraction, and
analyzed by 11% denaturing PAGE gel. Gels were dried, exposed in a
PhosphorImager cassette, and the screen was scanned in a Molecular Dynamics
PhosphorImager.

6. DNA ligation assay
The DNA ligation assay was performed in a 10 uL reaction. DNA
substrates (20 nM) were first incubated with or without protein Ku and/or XLF in
1X ligation reaction buffer (25 mM Tris-hydrochloride, pH 7.5, 75 mM NaCl, 72.5
mM KCl, 2 mM DTT, 0.025% Triton X-100, and 100 uM EDTA) supplemented with
10% PEG (MW>8,000D), 50 ug/mL BSA, and 5% glycerol at room temperature
for 15 min. The EDTA was used to eliminate effects of any possible trace divalent
cations, but is not necessary. Mg
2+
is added where specified above this low level
of EDTA. PEG improves the ligation efficiency substantially. The low level of

25
Triton X-100 is irrelevant to the ligation efficiency. Ligation was initiated by adding
100 uM of each dNTP, 10 mM MgCl
2
with different combinations of proteins as
indicated. Reactions were then incubated at 37°C for 30 min or 2.5 min as
indicated. After incubation, reactions were stopped, deproteinized with organic
extraction, and analyzed by 8% or 10% denaturing PAGE gel. Gels were dried,
exposed in a PhosphorImager cassette, and the screen was scanned in a
Molecular Dynamics PhosphorImager.

7. Sequencing of junctions of the ligation products
Dried gels were further exposed to Kodak films. After overnight exposure,
films were developed and then dimer ligation products (single-stranded because
they were being purified from a denaturing gel) were cut out of the gels by aligning
the gel with the film. Gel pieces were then soaked in appropriate amount (~30 uL)
of TE (10 mM Tris-hydrochloride, pH 8.0, 1 mM EDTA, pH 8.0) overnight.
Junctions were then amplified from the dimer ligation products by polymerase
chain reaction (PCR). PCR conditions were as follows: 95°C 3 min; 35 cycles of
94°C 45s, 53°C 1.25 min, 72°C 30s; 72°C 2 min. The sequences for the PCR
primers were: JG187 (5’-TGCTAGACTACTGGTCAAGC-3’), JG188
(5’-TGCATCCGTCAAGTAAGATG-3’). The size of PCR products was further
confirmed by 10% native PAGE gel and cloned into the Top TA cloning vector

26
pCR2.1 (Invitrogen, Calsbad, CA) according to the manufacturer’s instructions.
Plasmid was then transformed into E. coli, and individual clones were sequenced
on a Li-Cor sequencer (Li-Cor, Lincoln, NE) following the manufacturer’s
instructions. For Figure 4.2, reactions were re-run on an 8% native PAGE gel,
then the dimer ligation products (double-stranded because they were being
purified from a native gel) were cut out of the gels using the same method as
above. Dimer ligation products extracted from the gel pieces with TE were then
directly TA-cloned and sequenced without any PCR step.

8. Template dependent primer extension assay
The primer extension assay was performed in a 10 uL reaction.
Radioactively labeled DNA substrate (25 nM) was incubated with the indicated
amount of polymerase in 1X primer extension buffer (25 mM Tris-Cl, pH 8.0, 50
mM KCl, 5 mM MgCl
2
, 1 mM DTT) supplemented with 50 ug/mL of BSA and 50
uM of dNTP mix (50 uM each) at 37°C for 60 min. After incubation, reactions were
directly analyzed by 10% denaturing PAGE gel. Gels were dried, exposed in a
PhosphorImager cassette, and the screen was scanned in a Molecular Dynamics
PhosphorImager.

27
CHAPTER 3
TEMPLATE DEPENDENT AND TEMPLATE INDEPENDENT ADDITION BY
POLYMERASE MU

Abstract
Human DNA polymerase mu belongs to the DNA polymerase X family.
Along with its family member polymerase lambda, polymerase mu has been
suggested to function in NHEJ pathway. Both polymerase lambda and
polymerase mu possess N-terminal BRCT domain through which they are
recruited by Ku to the DSB site where gap fill-in is necessary. Here we find that
polymerase mu has both template dependent and template independent addition
capabilities on DSB end substrate under physiological conditions. Template
independent addition by polymerase mu prefers to add pyrimidines onto the DNA
ends. Furthermore, template independent addition onto the 3’ overhang of the
DNA substrates by polymerase mu provides terminal microhomology for ligation
by Ku and XRCC4:DNA ligase IV.

Introduction
Human DNA polymerase mu was identified in 2000 by Blanco lab
(Dominguez et al., 2000). It belongs to the DNA polymerase X family and shows

28
41% amino acid identity with one of its family members, TdT. Similar to TdT,
polymerase mu displays intrinsic template independent terminal
deoxynucleotidyltransferase activity, preferentially under Mn
2+
conditions.
However, a complementary template does dramatically improve the catalytic
efficiency of polymerase mu. Thus, polymerase mu is still regarded as a
DNA-dependent DNA polymerase. Interestingly, during nucleotide insertion with
its template dependent activity, polymerase mu presents low base discrimination
under either Mn
2+
or Mg
2+
conditions, with strong effect shown under Mn
2+

conditions (Dominguez et al., 2000). In addition, polymerase mu is able to insert
ribonucleotides opposite template strand with similar efficiency as of
deoxynucleotides. And XRCC4:DNA ligase IV readily joins those broken DNA
substrates with a single 3’ terminal ribonucleotide. Those special features provide
NHEJ an advantage during G1 cell cycle or in noncycling cells when the nuclear
dNTP pool in the cell is low (Nick McElhinny and Ramsden, 2003).
Family X DNA polymerases are low processing enzymes which include
polymerase beta, polymerase lambda, polymerase mu and TdT. Among them,
TdT has a restricted role in promoting antigen receptor diversity in lymphocytes
during V(D)J recombination, while polymerases lambda and mu have been
suggested to function in general NHEJ process to repair DNA DSBs. Both
polymerase lambda and polymerase mu possess N-terminal BRCT domain

29
through which they are recruited by Ku to the DSB site where gap fill-in is
necessary (Ma et al., 2004).
One group has proposed that polymerase mu can polymerize across a
discontinuous template strand, whereas polymerase lambda was not reported to
do so (Nick McElhinny et al., 2005). This ‘jumping’ from one DNA end to another
was proposed as a basis for why polymerase mu deficiency results in an altered
V(D)J recombination phenotype, where joining of two DNA ends with incompatible
3’ overhangs is suspected to be common (Schlissel, 1998). However, both the
polymerase lambda null mice and the polymerase mu null mice turn out to have
phenotypes in V(D)J recombination (Bertocci et al., 2003; Bertocci et al., 2006),
raising questions about models based on different abilities of polymerase mu and
polymerase lambda to synthesize across discontinuous templates.
Here we find that polymerase mu has both template dependent and
template independent addition capabilities on DSB end substrate under
physiological conditions. Template independent addition by polymerase mu
prefers to add pyrimidines onto the DNA ends. Furthermore, template
independent addition onto the 3’ overhang of the DNA substrates by polymerase
mu provides terminal microhomology for ligation by Ku and XRCC4:DNA ligase
IV.

30
Results
Polymerase mu has template independent polymerase activity under
physiological conditions and has a preference for pyrimidine addition
Previous work suggested that human polymerase mu and polymerase
lambda have template independent polymerase activity, but nearly all of this work
was carried out using Mn
2+
as the divalent cation (Dominguez et al., 2000;
Garcia-Diaz et al., 2000; Juarez et al., 2006; Ramadan et al., 2003; Ramadan et
al., 2004). Data for polymerase mu template independent polymerase activity
under Mg
2+
conditions were limited to a single-stranded DNA substrate
(Dominguez et al., 2000) or to substrates with long (10–20 nt) 5’ overhangs (Covo
et al., 2004). Those data are not applicable to the NHEJ scenario, which normally
generates 1 to 4 nts short overhangs. We were interested in testing whether
polymerase mu has template independent polymerase activity under
physiological conditions (Mg
2+
present) at dsDNA ends of the type subject to
NHEJ.
Our substrates for these studies consist of 73 bp dsDNA with an additional
2 nt overhang at each 3’ end (Figure 3.1A). To vary the end sequence, we had
one substrate with –AG on both ends, and the other with –TC on both ends. The
DNA substrates are 5’-radiolabeled with
32
P at one end, but the other DNA end
has a 5’ OH. We added polymerase mu to these substrates in free solution, and in

31
selected reactions we also added Ku and XRCC4:DNA ligase IV (Figure 3.1B).
After incubation to permit addition of nucleotides by polymerase mu, we analyzed
the products using denaturing PAGE.
We found that polymerase mu alone had little or no template independent
activity under these conditions (Figure 3.1B, lanes 2 and 12). But when Ku and
XRCC4:DNA ligase IV were present, we noted significant mononucleotide
addition to the 3’ overhanging ends (Figure 3.1B, lanes 7–10 and 17–20). On the
basis of single nucleotide iterations, polymerase mu appears to carry out
distributive synthesis on both substrates here. The presence of both Ku and
XRCC4:DNA ligase IV were important to achieve maximal polymerase mu
nucleotide addition (Figure 3.1C, lanes 9 and 10 versus 1–8; also Figure 3.1B).
This is consistent with the fact that Ku can bind to the BRCT domain of
polymerase mu (Ma et al., 2004), and that XRCC4:DNA ligase IV may provide
additional stability for the Ku:polymerase mu interaction (Mahajan et al., 2002).
Interestingly, the two types of DNA ends do not give identical results. Total
nucleotide addition to the –AG end is more efficient than to the –TC end. This may
reflect better initial binding of the polymerase mu to this terminal sequence. T is
not only the obvious preferred nucleotide for the –AG end (Figure 3.1B, lane 10),
but is seen on longer exposure to be the nucleotide associated with the longest
additions at the –TC end also (Figure 3.2B, lane 20). However, it is important to

32
note that all four dNTPs could be added to each of the two types of overhangs
(Figure 3.1B, lanes 7–10 and 17–20). Otherwise, one would expect C and T
addition only to the –AG end and G and A to the –TC end, and this is clearly not
the case. Therefore, polymerase mu has template independent activity under
physiological conditions.

33
Figure 3.1 Template independent polymerase activity of polymerase mu on
substrates in free solution.
(A) Two 73 bp substrates with 3’ overhangs were used to test for
polymerase mu template independent polymerase activity in free solution. An
asterisk indicates the position of the radioisotope label.
(B, C) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above each lane in a 10 uL reaction for 1 h at 37
o
C. After incubation,
reactions were deproteinized and analyzed using 11% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu, 25 nM, where X4-LIV
refers to XRCC4:DNA ligase IV. The specified dNTP was added to 100 uM. ‘dN’
means that all the four dNTPs (100 uM each) were included. No ATP was added,
unless specified. Template independent polymerase synthesis results in
extension of the radiolabeled strand, and hence, the more slowly moving species
located above the substrate band.

34
Figure 3.1, continued

35
Figure 3.2 Template independent polymerase activity of polymerase mu
provides short terminal microhomology for ligation.
(A) Two 73 bp substrates with 3’ overhangs were used to test for the
ligation by XRCC4: DNA ligase IV. An asterisk indicates the position of the
radioisotope label.
(B) This figure is from the same denaturing PAGE gel as Figure 3.1B. In
Figure 3.1B, we focused on the substrate addition part, while here we focused on
the ligation products, which are much higher on the gel. There is a crack on top of
the gel.
(C) Dimer products from the selected lanes were cut out of the gel,
extracted, and then PCR amplified, TA-cloned and sequenced. The junction
sequences for the ligatable strand are shown. Though 9 of 10 sequences from
lane 8 contained a CC junctional addition, 1 sequence contained a CT addition.
Deamination of dCTP to form dUTP presumably accounts for this one T.

36
Figure 3.2, continued

37
Polymerization by polymerase mu at free DNA ends is template independent
and not due to use of another DNA end as the source of a template strand
Polymerase mu also has well-documented template dependent activity
(Dominguez et al., 2000, Figure 3.3). To definitively rule out any possibility of
template dependent addition by polymerase mu, we immobilized the DNA at one
end to streptavidin–agarose beads using biotin. The DNA was used at a level
100- to 1000-fold below the binding capacity of the beads to ensure that the DNA
ends could not contact one another. Moreover, we showed that the DNA ends of
the bead-bound substrates could no longer be ligated, verifying the highly
dispersed distribution of the DNA substrate on the beads (Figure 3.5B).
Polymerase synthesis studies were conducted similar to those described in the
previous section, using the same types of DNA ends (Figure 3.4A). We still
observed template independent addition that cannot be explained by using
another DNA end as a template (Nick McElhinny et al., 2005). Specifically,
polymerase mu could add any of the four nucleotides to the –AG or to the –TC
ends (Figure 3.4B, lanes 2–5 and 9–12). Not surprisingly, the overall efficiency of
the reaction on the bead was lower. Therefore, to observe products, we increased
the amount of polymerase used. Yet we still observed that any of the four
nucleotides could be added. Therefore, polymerase mu can add nucleotides in a
template independent manner under physiological conditions to 3’ overhangs.

38
There are some interesting additional points to note. First, C and T are the
preferred nucleotide additions at both the –AG and the –TC ends (Figure 3.5B,
lanes 3 and 5 for the –AG ends and lanes 10 and 12 for the –TC ends). It is clear
that over 20 C and T nucleotides can be added to the free –AG ends. For the –TC
end, over 20 C nucleotides can be added, and addition of T also clearly occurs,
but not as efficiently as C. Hence, pyrimidines seem to be added better than
purines, regardless of the sequence of the end. The difference between the
number of nucleotides added at –AG and –TC ends may relate to how the
polymerase contacts the DNA at the double to single-strand transition region.
Second, nucleotide addition by polymerase mu alone without Ku and
XRCC4:DNA ligase IV is substantial for the immobilized substrates (Figure 3.4,
lanes 6 and 13). This may mean that immobilization of the DNA makes it easier
for polymerase mu to add nucleotides in a template independent manner, even
without the stabilizing influence of Ku and XRCC4:DNA ligase IV.
We did the same type of study for template independent addition to the free
end of bead-bound DNA substrates, but we replaced the dNTPs with the
corresponding ribonucleotides (NTPs). Previous work had shown that polymerase
mu can perform fill-in synthesis using NTPs (Nick McElhinny and Ramsden, 2003).
However, template independent synthesis with NTPs had not been reported. Not

39
surprisingly, like dNTPs, the corresponding NTPs can be added in a template
independent manner (Figure 3.6).
In summary, these immobilized substrate experiments demonstrate that
polymerase mu can carry out robust template independent synthesis under
physiological conditions, and the level of this activity suggests that this is the
dominant mode of addition at a free DNA end with a 3’ overhang.
We also studied human polymerase lambda, which previously was shown
to have template independent activity, but only in Mn
2+
solutions (Ramadan et al.,
2003). At equivalent molar amounts, polymerase lambda has a similar or even
slightly higher template dependent activity than polymerase mu (Figure 3.3).
However, when tested for template independent activity, we observed only a very
marginal level, regardless of addition of Ku or XRCC4:DNA ligase IV, and
regardless of whether the DNA substrate is free in solution or immobilized on
beads (data not shown).

40
Figure 3.3 Template dependent primer extension activity of polymerase mu
and polymerase lambda.
(A) A double-stranded DNA substrate with a 5’ overhang was designed to
test the template dependent primer extension of Pol X family polymerases. An
asterisk indicates the position of the radioisotope label.
(B) In each reaction, 25 nM substrate was incubated with the polymerase
in a 10 uL reaction for 1h at 37°C. After incubation, reactions were directly
analyzed by 10% denaturing PAGE. Protein concentrations varied as indicated.
50 uM of dNTP mix (50 uM each) was included to each reaction.

41
Figure 3.4 Template independent synthesis by polymerase mu on
immobilized DNA substrates distributed at low density on agarose beads.
(A) Streptavidin agarose beads were used to immobilize two 73 bp DNA
substrates with 3’ overhangs. ‘B’ designates the 3’-biotin group of the substrate.
An asterisk indicates the position of the radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above each lane in a 20 uL reaction for 1 h at 37
o
C. After incubation,
reactions were heated at 100
o
C for 5 min to disrupt the biotin–streptavidin
interaction, and then deproteinized and analyzed using 11% denaturing PAGE.
Protein concentrations: Ku, 50 nM; X4-LIV, 100 nM; polymerase mu, 1.25 uM;
dNTP, 5 mM. No ATP was added.

42
Figure 3.5 Template independent polymerase activity of polymerase mu on
immobilized DNA substrates.
(A) Streptavidin agarose beads were used to immobilize two 73bp DNA
substrates with 3’ overhangs. B designates the 3’ biotin group of the substrate. An
asterisk indicates the position of the radioisotope label.
(B) This image is simply a darker exposure of Figure 3.4B to illustrate the
multiple cycles of addition using dCTP and dTTP by polymerase mu. No ligation
products are seen even on this darker exposure, which documents that the bead
immobilization precludes any end-to-end interaction.

43
Figure 3.6 Ribonucleotide addition by polymerase mu on immobilized DNA
substrates.
(A) Diagram of immobilized substrates.
(B) The same substrates as in Figure 3.4A were used to test for
polymerase mu template independent polymerase activity with ribonucleotides. In
each reaction, 20 nM substrate was incubated with the protein(s) indicated above
each lane in a 20 uL reaction for 1h at 37°C. After incubation, reactions were
heated at 100°C for 5 min to disrupt the biotin-streptavidin interaction, and then
deproteinized and analyzed by 11% denaturing PAGE. Protein concentration:
polymerase mu, 1.25 uM. 500 uM of each dNTP/NTP was added to the reaction.
No Ku or XRCC4:DNA ligase IV was added in the reaction.

44
The template independent synthesis by polymerase mu provides the
microhomology for end ligaton by Ku and XRCC4:DNA ligase IV
Having gained a better understanding of the polymerization properties of
polymerase mu, we were interested in understanding the role of polymerase mu
in the ligation of the same types of DNA ends described above (Figure 3.7A). For
any given ligation reaction, duplex DNA substrates with 2 nt overhangs were used.
One 5’ end was radiolabeled with
32
P, and the other 5’ end was not ligatable
because it had a 5’ OH. To carry out the ligation reactions, we added Ku,
XRCC4:DNA ligase IV, dNTPs, and either polymerase mu or polymerase lambda.
We incubated reactions for 30 min, deproteinized, and analyzed with denaturing
PAGE.
Even when Ku, XRCC4:DNA ligase IV, and polymerase mu are all present,
we find that the ligation depends on the presence of specific dNTPs that could
potentially provide 1 or 2 bp of complementarity (Figure 3.7B, lanes 6, 8, and 9
versus 4, 5 and 7). To confirm this, we cut out the dimer species from the gel,
PCR amplified across the ligation junction, cloned into a TA cloning vector,
transformed bacteria, isolated the plasmid, and sequenced the individual ligation
junctions (Figure 3.7C). For reactions in which only dTTP is present, the junctions
only contain a T addition, which provides 1 bp of terminal microhomology. The
resulting single A:T bp is adequate to support the ligation. For reactions that

45
contain all four dNTPs (Figure 3.7B, lane 3), sequencing of the dimer product
shows junctions with a spectrum of additions, but predominantly a single T (Figure
3.7C, upper). As expected based on template independent synthesis at a DNA
end, the number of junctions with CT additions, which would provide 2 bp of
terminal microhomology, was considerably smaller. A few junctions illustrated
additional template independent addition which ended in CT, providing
complementarity, but which showed addition of other nucleotides 5’ to the CT.
Most of the added nucleotides were pyrimidines, illustrating the pyrimidine
preference noted earlier. Hence, in the presence of Ku and XRCC4:DNA ligase IV,
polymerase mu can support template independent addition at DNA ends, and the
resulting subset of ends that acquire complementarity can now be ligated.
Substrates with a –TC overhang can be ligated when dATP or dGTP are provided
(Figure 3.2B, lanes 17 and 19). When the dimer ligation products are sequenced,
the expected A or G nucleotides are present, consistent with the terminal
microhomology for joining being provided by template independent addition.
The above results indicate that 1 bp of annealing between two DNA ends is
sufficient to support ligation, even when the other strand remains in an unligatable
configuration. Given this, we reasoned that if we provide that one specific base
pair of terminal microhomology in the starting substrate, then the ligation should
proceed even without any polymerase present. We found that, indeed, this is the

46
case (Figure 3.8B, lane 4 on both panels). A substantial amount of dimer and
higher ligation multimers can be formed when the one DNA end overhang is
–AGT and is ligated to a DNA end with an incompatible 3’ overhang (Figure 3.8B,
right panel, lane 3). The bottom strand in this case would remain unligated for
three reasons: because there is no 5’ phosphate at the junction; there is a gap on
the bottom strand; and the right bottom strand has a 1 nt 3’ flap.
Surprisingly, even an –AGC 3’ overhang on the top strand can be ligated to
a GA– 3’ overhang end to form a small, but obvious amount of dimer (Figure 3.8B,
left panel, lane 4). This is unanticipated because the C of the –AGC must ligate
across a 1 nt gap to achieve ligation. The amount of ligation was lower than that
seen for the –AGT substrate (Figure 3.8B, compare left panel, lane 4 versus right
panel, lane 4), and seemingly, the explanation might be the difficulty in ligating
across the 1 nt gap. In fact, this is due to the steric hindrance over the overhang
sequences which will be discussed in detail in chapter five. The efficiency of the
ligation for the –AGT substrate explains the large number of products in which a T
is added when the overhang is –AG (Figure 3.7C, upper, junction sequence a
versus b–e). These studies confirm that random addition of a single
complementary nucleotide is sufficient to support ligation, and suggests that this
occurs even if this ligation must occur across a 1 nt gap.

47
When we tested a corresponding amount of polymerase lambda relative to
polymerase mu, as assessed by template dependent synthesis (Figure 3.3),
polymerase lambda supported only a low level of ligation (Figure 3.7B, lane 2).
We sequenced the small number of junctions formed, and these had T, CT, or GT
additions, consistent with a very low level of template independent addition, some
of which provided 1 bp of terminal microhomology.

48
Figure 3.7 Polymerase mu template independent polymerase activity
provides terminal microhomology for ligation by XRCC4:DNA ligase IV.
(A) The same substrate as in Figure 3.2A (left side) was tested for ligation.
Two alternative joining pathways are proposed below the substrate. From the
ligation patterns in (B) and Figure 3.9, we know that the first pathway is favored.
An asterisk indicates the position of the radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above each lane in a 10 uL reaction for 30 min at 37
o
C. After incubation,
reactions were deproteinized and analyzed using 8% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu or lambda, 25 nM.
dNTP (100 uM) was added to reactions, where indicated. ‘dN’ means all the four
dNTPs (100 uM each) were included. ATP (100 uM) was also added in indicated
reactions. ‘M’ indicates 50 bp DNA ladder. The dimer ligation product that results
from the joining of two substrate molecules is labeled. Joining of more than two
substrates results in trimer and higher-order species labeled as multimers.
(C) Dimer products from the selected lanes were cut out of the gel,
extracted, and then PCR amplified, TA cloned, and sequenced. The junction
sequences for the ligatable strand were provided. For lane 3, sequencing
information was collected and combined from three individual reactions. For lane

49
Figure 3.7, continued
8, two bands are apparent in the dimer product, but the longer product was not
among the four molecules sequenced.

50
Figure 3.7, continued

51
Figure 3.8 One base pair of terminal microhomology is sufficient for direct
ligation by XRCC4:DNA ligase IV.
(A) Two substrates with only 1 bp of terminal microhomology for ligation
were designed, based on the Figure 3.7A substrate, to test the direct ligation by
XRCC4:DNA ligase IV. Two alternative joining products are proposed below each
substrate. From both the ligation patterns in (B) and Figure 3.9, we know that the
upper product is favored over the lower product. An asterisk indicates the position
of the radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above in a 10 uL reaction for 30 min at 37
o
C. After incubation, reactions
were deproteinized and analyzed by 8% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu or lambda, 25 nM.
Twenty-five micromolar of each dNTP were in the reaction as indicated. ATP (100
uM) was also added in indicated reactions. ‘M’ indicates 50 bp DNA ladder.

52
Figure 3.9 Phosphate repulsion can decrease the ligation efficiency.
(A) The same substrate as in Figure 3.7A was tested for the phosphate
effect on ligation efficiency. The left substrate is exactly the same as in Figure
3.7A in which the bottom strand has a 5’ OH end. The bottom strand of the right
substrate was also phosphorylated on the 5’ end. Two alternative joining products
are proposed below each substrate. An asterisk indicates the position of the
radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above each lane in a 10 uL reaction for 30 min at 37°C. After incubation,
reactions were deproteinized and analyzed by 8% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu or lambda, 25 nM.
Each reaction contains all the four dNTPs (100 uM each). 100 uM of ATP was
also added in each reaction. “M” indicates 50 bp DNA ladder.

53
Figure 3.9, continued

54
Discussion
Polymerase mu template independent and template dependent addition
In this study, we have shown that polymerase mu can add nucleotides in a
template independent manner under physiological conditions. The duplex
substrates were immobilized on beads at a density that is 100 to 1000 fold lower
than the bead binding capacity, ensuring that synapsis of DNA ends is driven to a
negligible level (Yu and Lieber, 2000), and we have shown that ligation is no
longer detectable (Figure 3.5). The T, A, G, and C are all added at similar
efficiencies to –TC ends (Figure 3.4, right). A darker exposure shows that more
than 15 C or T nucleotides can be added (Figure 3.5).
Interestingly, the immobilized substrate with a –AG overhang also shows
addition of all four dNTPs at the DNA terminus, consistent with template
independent addition (Figure 3.4, left). As described above, a darker exposure
shows that runs of either C or T can exceed 15 nucleotides in length (Figure 3.5).
The similarity between the –TC and –AG ends in the propensity for C and T
addition indicates that there is a bias in favor of pyrimidines in the template
independent addition. Polymerase mu synthesis in Mn
2+
also favors pyrimidine
addition at the 3’ end of single-stranded DNA substrates (Dominguez et al., 2000).

55
When we compare polymerase mu polymerization at the DNA termini of
immobilized DNA fragments with that found for DNA substrates in free solution,
the results are indistinguishable for the ends terminating with a –TC 3’ overhang.
There is substantially more polymerase mu addition of T for the –AG
substrate in free solution than for the immobilized –AG substrate, even though
multiple rounds of T addition are clearly occurring even at the ends of the
immobilized –AG substrate as well. We do not yet know the basis for the
extremely efficient T addition, exceeding 40 nucleotides, but it might reflect a
combination of several factors: (a) the pyrimidine bias of polymerase mu template
independent activity; (b) some difference in the ability of polymerase mu to bind to
some overhang sequences versus others; and (c) the frequency of productive
encounters between polymerase mu and the DNA substrate. This last factor may
contribute to the difference between the immobilized and free –AG substrates,
whereas more than one factor may contribute to the difference between the free
–AG and free –TC substrates.
Km measurements for polymerase mu have been carried out using fill-in
synthesis substrates and show tighter binding of dATP and dGTP relative to dTTP
(Nick McElhinny and Ramsden, 2003). These measurements may not apply to the
template independent additions by polymerase mu, given its preference for use of
dCTP and dTTP.

56
Differences in template independent addition among polymerase mu,
polymerase lambda, and TdT
We observe only weak template independent addition by polymerase
lambda, whereas we observe that polymerase mu has substantial template
independent addition. Some structural differences between polymerase mu and
polymerase lambda have been suggested to be due, in part, to variation in a
region called loop 1 of these Pol X polymerases (Delarue et al., 2002; Juarez et
al., 2006; Nick McElhinny et al., 2005). Loop 1 may occupy part of the polymerase
catalytic region encountered by a template strand, thereby causing variation in the
balance of the template dependent and template independent modes.
Previous work suggested that polymerase mu could polymerize across a
discontinuous template strand, effectively jumping from one DNA end to another
(Nick McElhinny et al., 2005). The data supporting such a mechanism were based
on ddNTP utilization by polymerase mu. Recent studies from the same group
suggest that polymerase mu may work with other NHEJ factors like Ku and
XRCC4:DNA ligase IV to bridge the DNA ends together in order to perform this
special template dependent function (Davis et al., 2008). However, some of the
data regarding polymerase mu in NHEJ cannot be explained by this jumping
across discontinuous template strand model. For example, a mutation of H329 in
polymerase mu decreases template independent synthesis (without an effect on

57
template dependent synthesis) and simultaneously decreases NHEJ (Moon et al.,
2007). This supports the view, based on our data here, that polymerase mu adds
nucleotides template independently at 3’ overhangs (at junctions lacking any
terminal microhomology), thereby permitting annealing between the two DNA
ends. Hence, our data indicate a primary role for end annealing provided both by
the template independent activity of polymerase mu and its special ability to jump
or polymerize across a gap in the template strand when there is no
microhomology.
In vivo, pro-B cells derived from mice lacking both polymerase mu and TdT
expression, and still expressing polymerase lambda, have a very low but
detectable level of template independent addition at their Ig heavy chain junctions
(Bertocci et al., 2006), consisting mostly of pyrimidine addition, particularly T. The
low level of template independent addition by polymerase lambda that we see
may correspond to this.
It is interesting that the Ig heavy chain rearrangement involves polymerase
lambda and TdT, but not polymerase mu, whereas Ig light chain rearrangement
involves polymerase mu but neither polymerase lambda nor TdT (Bertocci et al.,
2006). Polymerase mu has robust template dependent and template independent
activity, whereas polymerase lambda has markedly greater template dependent
synthesis than template independent synthesis. Hence, the heavy chain

58
rearrangement involves two separate proteins that provide template dependent
(polymerase lambda) and template independent activity (TdT), whereas light
chain rearrangement involves polymerase mu, which has both activities in the
same polymerase.

59
CHAPTER 4
FLEXIBLE LIGATION CAPABILITY BY XRCC4:DNA LIGASE IV

Abstract
XRCC4 and DNA ligase IV form a complex that is essential for the repair of
all double-strand DNA breaks by the nonhomologous DNA end-joining (NHEJ)
pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate
two double-stranded DNA ends that have fully incompatible short 3’ overhang
configurations with no potential for base pairing. Moreover, at DNA ends that
share 1–4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1
nt. Ku can stimulate the joining, but is not essential when there is some terminal
annealing. Hence, annealing at sites of microhomology is very important, but the
flexibility of the ligase complex is paramount in nonhomologous DNA end joining.
In addition, XRCC4:DNA ligase IV has the distinctive ability to ligate poly-dT
single-stranded DNA and long dT overhangs in a Ku and XLF independent
manner, but not other homopolymeric DNA. The dT preference of the ligase is
interesting given the sequence bias of polymerase mu in NHEJ. These
observations provide an explanation for several in vivo observations that were
difficult to understand previously.

60
Introduction
In mammalian cells, DNA ligation proceeds through three consecutive
steps: 1) transfer of the AMP group from ATP to a lysine side chain of the ligase to
form an enzyme-adenylate complex; 2) transfer of the activated AMP group of the
DNA ligase-adenylate complex to the 5’ phosphate group of the substrate; 3)
formation of the phosphodiester bond in DNA and release of the AMP group. The
second and third steps usually occur as a concerted reaction. Till now, only three
DNA ligase families have been identified in mammalian cells: DNA ligase I, DNA
ligase III, and DNA ligase IV (Ellenberger and Tomkinson, 2008; Tomkinson et al.,
2006).
In NHEJ, XRCC4:DNA ligase IV complex is responsible for the final ligation
step, and it is considered to be the feature component for the NHEJ pathway
(Grawunder et al., 1997; Schar et al., 1997; Teo and Jackson, 1997; Tomkinson et
al., 2006; Wilson et al., 1997). XRCC4 facilitates DNA end joining by stabilizing
DNA ligase IV and stimulating its activity. XRCC4 interacts with DNA ligase IV
through the linker region between two BRCT domains on the C-terminal of ligase
IV, which is named “XRCC4 interacting region” (XIR) (Grawunder et al., 1998).
Recent crystal structure studies suggest that XIR is necessary for the interaction
between XRCC4 and DNA ligase IV, but additional residues from the C-terminal
distal BRCT domain are required to stabilize the interaction (Wu et al., 2009). Ku

61
improves the binding of the XRCC4:DNA ligase IV complex to DNA ends (Chen et
al., 2000; Nick McElhinny et al., 2000).
The active site in DNA ligase IV is lysine 273. Interestingly, about half of the
DNA ligase IV molecules in the highly biochemically purified XRCC4:DNA ligase
IV fraction are adenylated (Chen et al., 2000; Robins and Lindahl, 1996), thus
makes ATP unnecessary for in vitro biochemical reactions.
A fundamental question in NHEJ is the relative contribution of protein
factors in configuring the ends for ligation and the role of the intrinsic terminal
DNA sequence (Daley et al., 2005b). Here we find that XRCC4:DNA ligase IV
plus Ku can ligate several fully incompatible DNA end configurations that do not
share even 1 bp of terminal microhomology. Moreover, XRCC4:DNA ligase IV can
ligate across short gaps. Terminal annealing of 1–4 bp can obviate the need for
Ku, although it is often still stimulatory. As discussed in the previous chapter,
when confronted with incompatible DNA ends, polymerase mu can add
nucleotides in a template independent manner, thereby creating random
microhomology in a subset of DNA ends. Hence, the template independent
activity of polymerase mu and the remarkable flexibility of the ligase complex
permit a wide range of incompatible DNA ends to be joined via NHEJ.

62
Results
XRCC4:DNA ligase IV can ligate across gaps
In the last chapter, we already observed that XRCC4:DNA ligase IV seems
able to ligate across a gap (Figure 3.8B, left panel, lane 4). To test formally
whether XRCC4:DNA ligase IV could ligate across a gap, we examined substrate
ligation by this ligase complex with or without other components (Figure 4.1). The
XRCC4:DNA ligase IV complex alone could ligate a 4 bp terminal microhomology
with a 1 nt gap in the ligatable strand (Figure 4.1B, left panel, lane 2). This ligase
complex could also ligate across a 1 nt gap using a 2 bp terminal microhomology
(Figure 4.1B, right panel, lane 2 and Figure 4.2B, lane 4). Sequencing of junctions
confirmed the ligation across gaps (Figure 4.1C and Figure 4.2C). Hence, the
XRCC4:DNA ligase IV can support ligation across gaps in the absence of other
proteins.
Ku stimulated the ligation across a gap (Figure 4.1B, left panel, lane 3, and
right panel, lanes 4 and 5). Addition of polymerase lambda or polymerase mu
further stimulated the ligation (Figure 4.1B, left panel, lanes 4–7), but this was
because addition of T occurred (sometimes along with additional nucleotides), as
documented by sequencing (Figure 4.1C, left, sequences from lanes 4 to 7, or
right, sequences from lanes 7 and 8; Figure 4.2C).

63
The rate of ligation of ends that have gaps adjacent to 2 bp of terminal
microhomology is roughly 10 times as slow as the rate of ligation of a nick
adjacent to a 4 bp block of terminal microhomology (Figure 4.3).

64
Figure 4.1 XRCC4:DNA ligase IV can ligate over a gap.
(A) Two substrates with different lengths of terminal microhomology for
ligation were designed to test the direct ligation over a gap by XRCC4:DNA ligase
IV. There is a one-nucleotide gap on the ligatable strand. Only the favored joining
product is shown under each substrate. An asterisk indicates the position of the
radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above in a 10 uL reaction for 30 min at 37
o
C. After incubation, reactions
were deproteinized and analyzed by 8% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu or lambda, 25 nM.
Twenty-five micromolars of each dNTP were added to the reaction as indicated.
All the reactions include 100 uM of ATP. ‘M’ indicates 50 bp DNA ladder.
(C) Dimer products from the selected lanes were cut out of the gel,
extracted, and then PCR amplified, TA-cloned, and sequenced. The junction
sequences for the ligatable strand were provided.

65
Figure 4.1, continued

66
Figure 4.2 XRCC4:DNA ligase IV and Ku can ligate over a gap.
(A) Two 57 bp substrates with 3’ overhangs were tested for ligation in the
same reaction. One substrate (JG171/172) has 5’ OH on both strands. The top
strand from the other substrate (JG*173/174) was radioactively labeled on the 5’
end. We note there are at least four joining pathways for the dimer product.
However, the proposed joining pathway under the substrate should be favored
due to homology on the end for ligation. The backbone (central) sequences (the
portion drawn as a top and bottom strand line) for the left duplex is different from
the backbone sequence for the right duplex.
(B) In each reaction, 10 nM unlabeled substrate and 10 nM labeled
substrate were incubated with the protein(s) indicated above in a 10 uL reaction
for 30 min at 37°C. After incubation, reactions were deproteinized and analyzed
by 8% denaturing PAGE. Protein concentrations: Ku, 25 nM; X4-LIV, 50 nM;
polymerase mu or lambda, 25 nM. Each reaction contains all the four dNTPs (25
uM each). 100 uM of ATP was also added in each reaction.
(C) The same reactions as in (B) were run on an 8% native PAGE. Then
dimer product from lane 4 (a reaction that included only XRCC4:DNA ligase IV
and Ku) was cut out of the gel, extracted, and then directly TA-cloned and
sequenced. The junction sequences for the ligatable strand were provided.

67
Figure 4.2, continued

68
Figure 4.3 Time course of ligation of a nick, a 1 nt-gap, and a fully
incompatible DNA end substrate by XRCC4:DNA ligase IV and Ku.
(A) JG*161/162 is a substrate with a nick on the ligatable strand.
JG*163/166 is a substrate with a 1 nt gap on the ligatable strand and with a two
base pair terminal microhomology for ligation. JG*163/186 is a substrate with fully
incompatible ends. A star indicates the position of the radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above the gel in a 10 uL reaction for the indicated time at 37°C. After
incubation, reactions were deproteinized and analyzed by 8% denaturing PAGE.
Protein concentrations: Ku, 25 nM; X4-LIV, 50 nM. “M” indicates 50bp DNA
ladder.
(C) Ratio of ligation products versus total reaction substrate was quantified
and provided.

69
Figure 4.3, continued

70
XRCC4:DNA ligase IV can ligate fully incompatible DNA ends
We were interested in the minimal amount of terminal microhomology
needed to support ligation (Figure 4.4A). We found that, in some cases, Ku and
XRCC4:DNA ligase IV were sufficient to complete the ligation of entirely
incompatible DNA ends (Figure 4.4B, lanes 4 and 5 versus 1–3). Sequences of
the dimer species confirmed that the ligation occurred with no alteration of either
DNA end (see Figure 4.4C, sequences from the dimer ligation product in lanes 4
and 5).
In addition to the ligation of this incompatible DNA end pair, we also
observed that a DNA end with a 3’ overhanging A could be ligated to an end with
a 3’ overhanging –AGC (data not shown). Another example of the ligation of two
incompatible ends is the joining of two DNA ends, each with a 3’ A overhang
(Figure 4.5). Hence, Ku and XRCC4:DNA ligase IV can ligate a subset of fully
incompatible ends that do not share even 1 bp of terminal microhomology.
The rate of ligation of DNA ends that are incompatible with no terminal
microhomology is obviously much slower than for ends that are stabilized by two
terminal base pairs or four terminal base pairs plus stacking (Figure 4.3). This is
not surprising given the stabilization and alignment contributed by such terminal
blocks of microhomology.

71
Figure 4.4 XRCC4:DNA ligase IV and Ku can ligate fully incompatible DNA
ends (1).
(A) A substrate without any homology for ligation was designed on the
basis of the second substrate in Figure 4.1A to test the ligation with XRCC4:DNA
ligase IV. Two alternative joining pathways are proposed next to the substrate. An
asterisk indicates the position of the radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above in a 10 uL reaction for 30 min at 37
o
C. After incubation, reactions
were deproteinized and analyzed by 8% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu or lambda, 25 nM.
Twenty-five micromolars of each dNTP were added to the reaction as indicated.
All the reactions include 100 uM of ATP. ‘M’ indicates 50 bp DNA ladder. In lane 7,
the band below the dimer product is most likely the hairpin structure of the dimer
product that is ligated in the manner we proposed in (A), second product.
(C) Dimer products from the selected lanes were cut out of the gel,
extracted, and then PCR amplified, TA-cloned, and sequenced. The junction
sequences for the ligatable strand were provided.

72
Figure 4.4, continued

73
Figure 4.5 XRCC4:DNA ligase IV and Ku can ligate fully incompatible DNA
ends (2).
(A) A substrate without any homology for ligation was tested for the direct
ligation by XRCC4:DNA ligase IV and Ku. Two alternative joining pathways are
proposed under the substrate. An asterisk indicates the position of the
radioisotope label.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above each lane in a 10 uL reaction for 30 min at 37°C. After incubation,
reactions were deproteinized and analyzed by 8% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu or lambda, 25 nM.
Each reaction contains all the four dNTPs (25 uM each). 100 uM of ATP was also
added in each reaction.
(C) Dimer products from the selected lanes were cut out of the gel,
extracted, and then PCR amplified, TA-cloned and sequenced. The junction
sequences for the ligatable strand are listed.

74
Figure 4.5, continued

75
XRCC4:DNA ligase IV is distinctive in those special ligation capabilities
How specific is the ability of XRCC4:DNA ligase IV to ligate across gaps at
DNA ends and to ligate incompatible DNA ends? We were interested in
comparing ligation of various DNA ends by equivalent levels of activity of T4 DNA
ligase, human DNA ligase I, DNA ligase III, and DNA ligase IV (plus XRCC4 and
Ku). We used a nicked duplex oligonucleotide substrate to establish equal activity
levels of each of these four ligases. We then compared the ligation of four
different substrate end configurations (Figure 4.6). The ligation of a nick adjacent
to a 4 bp block of terminal microhomology is similar for all four ligases (Figure
4.6B, lanes 2–5). However, XRCC4:DNA ligase IV is distinctly better at ligating
end pairs that have a 1 nt gap adjacent to a 4 bp block of microhomology (Figure
4.6B, lanes 7–10) or adjacent to a 2 bp block of microhomology (Figure 4.6C,
lanes 2–5). For incompatible DNA ends with no microhomology, XRCC4:DNA
ligase IV ligation was detectable, whereas ligation by the other ligases was
undetectable (Figure 4.6C, lanes 7–10). Ku has no effect on the activity of DNA
ligase I or III in these ligations (Figure 4.6B–D).
Although many pairs of ends lacking microhomology can be ligated by Ku
and XRCC4:DNA ligase IV, this was not universal. We found that when both ends
had 3’ overhangs of two incompatible nucleotides, then joining did not occur (data
not shown). In summary, pairs of DNA ends that have very short, fully

76
incompatible 3’ overhangs can be ligated by Ku and XRCC4:DNA ligase IV, but
longer overhangs cannot be ligated.

77
Figure 4.6 Ligase activity comparison with DNA double-strand break
substrates.
(A) JG*161/162 is a substrate with a nick on the ligatable strand.
JG*162/161 is a substrate with a 1 nt gap on the ligatable strand and has 4 bp of
terminal microhomology for ligation. JG*163/166 is a substrate with a 1 nt gap on
the ligatable strand and has a 2 bp terminal microhomology for ligation.
JG*163/186 is a substrate with fully incompatible ends for ligation. A star indicates
the position of the radioisotope label.
(B), (C), and (D), in each reaction, 20 nM substrate was incubated with the
protein(s) indicated above in a 10 ul reaction for 30 min at 37
o
C. After incubation,
reactions were deproteinized and analyzed by 8% denaturing PAGE. Protein
concentrations: Ku, 25 nM; X4-LIV, 50 nM; human ligase I, human ligase III, and
T4 DNA ligase were first normalized to the same activity of 50 nM X4-LIV using a
single-strand nick substrate, and then used in equivalent amounts of activity for
the reactions shown. One millimolar of ATP was also added in specified reactions.
‘M’ indicates a 50 bp DNA ladder. Ligase abbreviations: I, human ligase I; III,
human ligase III-alpha; IV, human XRCC4:DNA ligase IV; T4, T4 DNA ligase.

78
Figure 4.6, continued

79
XRCC4:DNA ligase IV can ligate single-stranded DNA
In our analysis of incompatible DNA end joining, we examined
progressively longer overhangs (up to 15 nt poly-dT overhangs). For comparison,
we tested for the ligation of poly-dT as single-stranded DNA. We were surprised
to detect a ladder of ligation products exceeding 6 unit molecules in length for the
poly-dT substrate (Figure 4.7). Notably, poly-dA,-dG,-dC and -rU were not
ligatable (data not shown). Moreover, a single terminal dT at the 5’ or 3’ end of the
single-strand was not sufficient to permit ligation. Five dT nucleotides at both the
5’ and 3’ end were readily ligated, regardless of the internal sequence (data not
shown). Neither Ku nor XLF could stimulate this single-strand ligation, probably
because Ku does not bind single-stranded DNA and XLF binds it very inefficiently
(Lu et al., 2007). In related studies, we have also observed ligation of a DNA
duplex with a long poly-dT overhang to a single-stranded poly dT molecule
(Figure 4.8, lanes 2 and 3) and ligation of two duplex molecules, one with a long 3’
dT overhang and the other with a long 5’ dT overhang (lanes 4–8). Hence, the
XRCC4:DNA ligase IV complex has remarkable flexibility to ligate single-stranded
DNA and long 5’ or 3’ overhangs. Yet the interactions of the single-stranded
substrate near the active site of the ligase complex may require more than one dT
nucleotide near the 5’ and the 3’ end.

80
Figure 4.7 Single-stranded DNA can be ligated by XRCC4:DNA ligase IV.
Single-stranded 30 dT substrate (JG*169) was tested for direct ligation by
XRCC4:DNA ligase IV. In each reaction, 50 nM substrate was incubated with
different amounts of XRCC4:DNA ligase IV as indicated above each lane in a 10
ul reaction for 30 min at 37
o
C. After incubation, reactions were deproteinized and
analyzed by 10% denaturing PAGE. ‘M’ indicates 50 bp DNA ladder.
Quantification shows that about 0.1% of the substrate is converted to the ligated
products in lanes 6–8. No ATP is present in the incubations, consistent with the
pre-charged status of DNA ligase IV.

81
Figure 4.8 Long dT overhangs can be directly ligated by XRCC4:DNA ligase
IV.
(A) Sequences for single-stranded dT substrates and long dT overhang
substrates are shown.
(B) Different combinations of substrates were tested for the ligation by
XRCC4:DNA ligase IV with or without Ku and/or XLF. In each reaction, 20 nM
double-stranded substrate with or without 50 nM single-stranded substrate were
incubated with different combinations of proteins as indicated above each lane in a
10 uL reaction for 30 min at 37
o
C. After incubation, reactions were deproteinized
and analyzed by 10% denaturing PAGE gel.

82
Discussion
Ligation of incompatible DNA ends by Ku and XRCC4:DNA ligase IV
Ligation without any terminal microhomology markedly expands the role of
Ku and XRCC4:DNA ligase IV in NHEJ. Previously, we and others assumed that
nucleases and polymerases were essential to bring two DNA ends to a point
where each strand would have a ligatable nick. Although nucleases and
polymerases markedly expand the number of ways in which two DNA ends can
be ligated, and thereby improve the efficiency of ligation, we now see that many
simple overhang combinations may be joined with neither a nuclease nor a
polymerase (Figure 4.9).
These biochemical findings fit well with important recent in vivo findings.
First, after ionizing radiation, the large majority of DSBs are thought to be
incompatible. Recently, it was suggested that only a subset of the DNA ends
require the endonucleolytic activity provided by the Artemis:DNA-PKcs complex
(Riballo et al., 2004). This observation can now be understood in light of the
findings here that Ku and XRCC4:DNA ligase IV may ligate many of the
incompatible DNA end configurations generated by the ionizing radiation.
Second, cells from mice fully deficient for both polymerase mu and
polymerase lambda are not sensitive to ionizing radiation (Bertocci et al., 2006).
We and others had previously assumed that this was because of the possible

83
involvement of other polymerases (Wilson and Lieber, 1999). However, the
finding that Ku and XRCC4:DNA ligase IV can ligate many incompatible DNA
ends without a polymerase provides one clear biochemical basis for why the
polymerase mu/polymerase lambda double null cells would not be sensitive to
ionizing radiation.
Third, the template independent addition by polymerase mu at short 3’
overhangs under physiological conditions finally provides an explanation for a
very puzzling aspect of data from V(D)J recombination junctions in TdT null mice.
Such mice can have template independent addition at nearly 5% of their V(D)J
junctions (Bertocci et al., 2006; Gilfillan et al., 1993; Komori et al., 1993). The
source of these template independent additions has been unclear. The template
independent addition by polymerase mu (and perhaps a much lower level by
polymerase lambda) can now be seen to explain these in vivo observations.
Incompatible DNA ends with 3’ overhangs are perhaps the most important
type of incompatible configuration in the repair of DSBs by NHEJ, because other
configurations can be ligated more simply. For example, fill-in synthesis by any of
a number of polymerases, including polymerase mu and polymerase lambda, can
convert 5’ overhangs to blunt configurations, and we previously showed that
XRCC4:DNA ligase IV can ligate blunt ends (Grawunder et al., 1997; Gu et al.,
2007b). Moreover, 5’ overhangs can be endonucleolytically cleaved to a blunt

84
configuration by Artemis:DNA-PKcs or exonucleolytically by Artemis alone (Ma et
al., 2002). In contrast, 3’ overhangs (or hairpins in V(D)J recombination) are
cleaved by Artemis:DNA-PKcs in a manner that typically leaves a short residual 3’
overhang, which cannot be readily filled in by a polymerase (Ma et al., 2002).
Hence, direct ligation of a subset of short incompatible 3’ overhangs by Ku and
the ligase complex is important. The only alternative manner of joining is to carry
out template independent addition, which we have demonstrated in the previous
chapter for polymerase mu and has been previously demonstrated for TdT, in the
case of V(D)J recombination (Ramadan et al., 2004).
Prokaryotic NHEJ systems appear to rely primarily on Ku and a ligase
activity with nuclease or polymerases playing roles for only specific types of DNA
end configurations (Della et al., 2004; Gong et al., 2005). In yeast NHEJ, Ku and
the Lif1:DNA ligase IV complex play major roles in NHEJ, whereas POL4 plays a
discernable, but nonessential role (Wilson and Lieber, 1999). As is the case for
prokaryotes, a nuclease may be required for only a subset of yeast NHEJ events
(Daley et al., 2005b; Downs and Jackson, 2004). Therefore, the ability to handle a
diverse set of overhangs using Ku and a ligase complex may have its origins early
in evolution, with the polymerases and nucleases contributing to subsets of DNA
end configurations to improve joining in selected cases.

85
Figure 4.9 Function of XRCC4:DNA ligase IV in ligating incompatible DNA
ends.
(A) Ku and XRCC4:DNA ligase IV can ligate fully incompatible DNA ends.
XRCC4:DNA ligase IV randomly bind to one DNA end, but this interaction is less
stable than if Ku is also bound. Ku may stimulate the interaction by increasing the
occupancy time of XRCC4:DNA ligase IV at the DNA end. When another DNA end
comes close to this complex, XRCC4:DNA ligase IV binds it and ligates the two
ends.
(B) XRCC4:DNA ligase IV alone can ligate across a gap. XRCC4:DNA
ligase IV randomly binds to one DNA end (although this interaction is not as stable
as when Ku is present). The 2 bp of terminal microhomology between the DNA
ends increases the chance for XRCC4:DNA ligase IV to ligate.
(C) Template independent polymerase activity of polymerase mu creates
terminal microhomology for ligation by XRCC4:DNA ligase IV. Polymerase mu can
add nucleotides to the DNA end in its template independent mode. A 1 bp terminal
microhomology between the DNA ends (‘t’ in this example) permits annealing of
the ends and improves the efficiency of ligation.

86
Figure 4.9, continued

87
Ligation across gaps by XRCC4:DNA ligase IV
XRCC4:DNA ligase IV is distinctive in its ability to ligate across gaps at
DNA ends. Studies of ligation by T4 DNA ligase across 1 nt gaps within duplex
DNA did not show any data on incompatible DNA end ligation, which is the type
shown here (Goffin et al., 1987). The manner in which XRCC4:DNA ligase IV
binds the substrate must be quite flexible to tolerate not only the normal nick
ligation, but also ligation across 1 nt gaps. The flexibility at the junction could arise
from flexibility in the position of the 3’ OH and the 5’ P at the nick. Alternatively,
the nucleotides across from the gap on the anti-parallel strand could be forced
into an extrahelical position. Obviously, there are many variations between these
two extremes, which are all equally plausible, given that the junctional DNA is
unlikely to be in standard B-DNA conformation. The recent cryo-EM studies using
Ku and DNA-PKcs suggested that the two DNA ends might be off-set (Spagnolo
et al., 2006). Some degree of off-set or alternative nonlinear alignment of the ends
(or at least tolerance for such angles) could also facilitate ligation across gaps.

Differences in template dependent addition by polymerase mu versus
polymerase lambda
Polymerase mu can improve the joining efficiency of some pairs of ends by
XRCC4:DNA ligase IV or Ku and XRCC4:DNA ligase IV (Figures 3.7 and 3.8, and

88
Chapter 4). Sequencing shows that a subset of such junctions show direct joining
without nucleotide addition, but most show some nucleotide addition, and this
addition provides the additional microhomology, which is the basis for the
increased joining efficiency.
For some pairs of DNA ends, polymerase lambda does not fill gaps as
efficiently as polymerase mu. For example, in Figure 4.4, lane 6, we see no
addition by polymerase lambda (no T added). But polymerase mu does
sometimes add a T or a GT. Part of this difference may be compensated by an
increase in the concentration of the polymerase. This is illustrated in Figure 4.1B,
right panel, where polymerase lambda does not fill in at the concentration of
polymerase lambda shown, but when we increase the concentration of
polymerase lambda five-fold, we do see some ends with addition (Figure 4.1).
This may account for in vivo observations of human polymerase mu and
polymerase lambda in S. cerevisiae deficient for POL4 (Daley et al., 2005a). The
stronger ability of polymerase mu versus polymerase lambda could account for
joining events where polymerase mu fills in gaps (after removal of unpaired
regions), whereas polymerase lambda fails to do so (Daley et al., 2005a).

89
The synapsis of DNA ends for ligation by XRCC4:DNA ligase IV
We find here that some pairs of incompatible DNA ends can be ligated with
only XRCC4:DNA ligase IV, and others either require Ku to improve the efficiency
of joining or are stimulated by Ku. Ku is known to improve the binding of
XRCC4:DNA ligase IV to DNA ends (Chen et al., 2000; Nick McElhinny et al.,
2000), and the stimulatory effect of Ku for the joining of some pairs of ends may
simply reflect this. One could wonder if Ku might actually be the synapsis factor,
as was suggested by previous work using purified Ku and DNA (Ramsden and
Gellert, 1998). However, synapsis activity of purified Ku has not been seen by
others (DeFazio et al., 2002), nor has a significant ligation stimulation of ligase I
and III by Ku (Figure 4.6).
Moreover, it is quite clear that Ku becomes dispensable when there is
terminal annealing of partially compatible DNA ends. The more the annealing
occurs, the more negligible the effect of Ku (Figures 4.1A, B and 4.4 show the
progression from 0, 2, and 4 bp of microhomology). Neither 2 nor 4 bp of terminal
annealing would be sufficient to provide a stable synapsis during the 37
o
C
incubations here. Rather, XRCC4:DNA ligase IV appears adequate to provide
synapsis without Ku, and terminal microhomology may contribute to the overall
stability of the two ends within the XRCC4:DNA ligase IV complex. When there is
no terminal microhomology, the addition of Ku may stimulate joining by increasing

90
the occupancy time of XRCC4:DNA ligase IV on either DNA end. Longer
occupancy time by XRCC4:DNA ligase IV at either end would then give more
opportunity for the partner DNA end to be encountered, bound, and the two ends
ligated. However, formal equilibrium and kinetic studies will be needed to discern
the precise contribution of each protein and the extent of end annealing to the
overall stability of the junctional complex.

Single-stranded DNA ligation
The single-stranded DNA ligation is noteworthy among DNA ligases. T4
RNA ligase has the ability to ligate single-stranded DNA (Brennan et al., 1983;
Snopek et al., 1976). Among DNA ligases, T4 DNA ligase has been reported to
ligate single-stranded DNA at a level that can only be detected by PCR; however,
the possibility of fold-back at the DNA termini (double-strandedness) was not
ruled out in that study, making even this trace level of activity uncertain (Kuhn and
Frank-Kamenetskii, 2005). Therefore, the single-stranded DNA ligation by
XRCC4:DNA ligase IV is quite remarkable, especially given that this ligase is also
capable of acting on a variety of duplex DNA end configurations. This suggests
that the mode of binding by this ligase to the substrate DNA is quite flexible,
accommodating either two single strands or two duplex strands.

91
CHAPTER 5
BIOCHEMICAL FUNCTION OF XLF (CERNUNNOS) IN NHEJ

Abstract
The double-strand DNA break repair pathway, nonhomologous DNA end
joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and
ligase activities. Here we find that the joining of ends by XRCC4:DNA ligase IV is
markedly influenced by the terminal sequence, and a steric hindrance model can
account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA
ends and compatible DNA ends at physiological concentrations of Mg
2+
, but only
of incompatible DNA ends at higher concentrations of Mg
2+
, suggesting charge
neutralization between the two DNA ends within the ligase complex.

Introduction
Since all the necessary enzymatic activities (nuclease, polymerase and
ligase) for NHEJ pathway have been identified and NHEJ pathway has also been
successfully biochemically reconstituted with all those known components (Ma et
al., 2004), people in the field generally regard this pathway is complete. Until 2003,
Oettinger lab derived 2BN cells from a patient with radiosensitivity and severe
combined immunodeficiency (Dai et al., 2003). They claimed that NHEJ requires

92
an additional factor since NHEJ deficiency in 2BN cells can not be ascribed to any
of the known NHEJ factors.
Three years later, two research groups independently identified this
missing component for NHEJ (Ahnesorg et al., 2006; Buck et al., 2006). One
group started from looking for XRCC4 interaction factors with yeast two-hybrid
screening, and identified a novel protein which displays structural similarity to
XRCC4, thus named as “XRCC4-like-factor” (XLF). In addition, XLF rescued the
NHEJ deficiency in 2BN cells (Ahnesorg et al., 2006). The other group started
with a few patients with a new syndrome as combined growth retardation,
microcephaly and immunodeficiency. Cells derived from those patients display
increased radiosensitivity, defective V(D)J recombination and impaired DNA
ligation, which indicates a defect in NHEJ process. Finally, they identified this
mutated protein in those patients with a cDNA functional complementation cloning
strategy. This new protein, they named it as “Cernunnos” (Buck et al., 2006). It is
the same protein as XLF.
However, although XLF represents a new factor in NHEJ, its biochemical
function in the NHEJ pathway is still unknown. Based on its structural similarity
with XRCC4, and its direct interaction with XRCC4 and DNA ligase IV, it has been
proposed to function in the final ligation step and may represent the third
component in the ligase complex.

93
Here we tested the biochemical function of XLF with our in vitro NHEJ
system. And we find that XLF (Cernunnos) stimulates the joining of both
incompatible DNA ends and compatible DNA ends at physiological concentrations
of Mg
2+
, but only of incompatible DNA ends at higher concentrations of Mg
2+
,
suggesting charge neutralization between the two DNA ends within the ligase
complex. In addition, we find that the joining of ends by XRCC4:DNA ligase IV is
markedly influenced by the terminal sequence, and a steric hindrance model can
account for this.

Results
Influence of terminal DNA sequence on ligation by XRCC4:DNA ligase IV
and stimulation by XLF
We have found that there is a wide variation in the efficiency of DNA end
ligation by XRCC4:DNA ligase IV due to minor variations in the DNA end
sequences. For example, the end sequences illustrated in Figure 5.1B, left panel
(–ACC3’ joined to 3’ GGA–) are joined well (on the top strand) by Ku plus
XRCC4:DNA ligase IV (lane 1), and this is only slightly increased by the addition
of polymerase mu (lane 2). However, the bottom strand of those same DNA ends
is not ligated well (lane 3), unless polymerase mu is present to fill in the 1 nt gap
(lane 4). This illustrates that the two strands of the same pair of DNA ends are not

94
ligated with equal efficiency by XRCC4:DNA ligase IV and some aspect of the
sequence at the two ligation points determines this difference. (Note that when we
test the top strand for ligation, the bottom strand has an unligatable 5’OH at the
junction. Likewise, when we test the bottom strand for ligation, the top strand has
an unligatable 5’OH at the junction.)
There are hundreds of variations of the two partially complementary DNA
ends of Figure 5.1A, and we have not analyzed all of them. However, a 1 nt
change in the complementary portion, such that the ends are –AGC3’ and 3’
CGA– now results in a substantial reduction (60-fold) in the ability of Ku plus
XRCC4:DNA ligase IV to join the top strand of these ends (Figure 5.1B, right
panel, lanes 3 versus 8). DNA sequence analysis confirmed the junctional
sequence. These results illustrate that 1 nt change in otherwise identical
overhangs can markedly affect the joining.
Importantly, XLF is able to permit joining to a much greater level for a pair
of inefficiently joined ends (Figure 5.1B, right panel, lanes 3 versus 4). In contrast,
ends joined efficiently are not stimulated further by XLF (lanes 8 versus 9).
[Assays done using shorter incubation times and less ligase rule out the
possibility that the lack of stimulation is due to the reaction reaching a plateau
(data not shown).] This raises the possibility that base pairings (2 bp here) that
are accommodated well by the ligase complex are sufficient to stabilize those

95
ends in a manner that XLF cannot improve upon, whereas more weakly
accommodated end sequences benefit from XLF. The following sections describe
studies to further explore both the sequence effects on ligation and the XLF
effects on ligation efficiency.

96
Figure 5.1 DNA substrates with a gap are ligated by XRCC4:DNA ligase IV in
a sequence dependent manner.
(A) Three 73 bp substrates with different 3’ overhangs were designed and
tested for the efficiency of ligation over a gap by XRCC4:DNA ligase IV. A star
indicates the position of the radioisotope label. JG*163/166 and JG*166/163 are
the same duplex DNA substrate but with one strand labeled or the other. In the
ligations, a dimer, e.g. involves two molecules of the same species joined head to
tail.
(B) In each reaction, 20 nM substrate was incubated with the protein(s)
indicated above the lane in a 10 ul reaction for 30 min at 37
o
C. After incubation,
reactions were deproteinized and analyzed by 8% denaturing PAGE. Protein
concentrations are as follows: Ku, 25 nM; X4-LIV, 50 nM; polymerase mu, 25 nM;
T4 DNA ligase, 120 nM; XLF, 100 nM. Twenty-five micromolar of each dNTP was
added to the reactions where polymerase mu was present. Hundred micromolar
of ATP was also added in each reaction shown in the left panel, and 1mM of ATP
was added to the reactions where T4 DNA ligase was present. ‘M’ indicates 50 bp
DNA ladder. Ligation products were quantified, and ligation efficiencies were
provided under each lane. DNA sequence analysis confirmed that the junctional
sequences conformed to those shown in A. Note that when the top strand is
assayed for ligation, the bottom strand has an unligatable 5’ OH at the junction,

97
Figure 5.1, continued
and likewise when the bottom strand is assayed for ligation, the top strand has an
unligatable 5’ OH at the junction.

98
Figure 5.1, continued

99
The relative efficiency of DNA end joining conforms to a model invoking
steric constraints on the ends within the ligase complex
For ligation with XRCC4:DNA ligase IV and Ku (with or without XLF), we
observed a certain pattern of ligation efficiencies for pairs of ends with partial
complementarity (2 bp of terminal microhomology with a 1 nt gap on each strand).
After annealing at any chance terminal microhomologies, the order of ligation
efficiencies for the various pairs of DNA ends fits a pattern most consistent with
some steric limitations due to purines at positions 2 and 3 nt from one 3’ end
being in conflict with purines on the anti-parallel strand, but at positions that are 1
nt shifted from the initial base pairing. The ligation efficiency is optimal when the
purine:purine conflict (R:R) is minimal within a slanted 2 nt region covering each
of the two overhangs (Figure 5.1A; see Figure 5.3 for a diagram of the model).
In order to test this model, we created many of the end pair configurations
for 3 bp 3’ overhangs that share 2 bp of terminal microhomology. Among the 11
substrate pairs created, nearly the entire set conformed to the model (Figure 5.2B
and Figure 5.1; see model in Figure 5.3). Specifically, the substrates with the least
R:R conflict within the 2 nt region are most efficiently ligated (Figure 5.1B, right
panel, lane 8 and Figure 5.2B, lane 13). The substrates with the most R:R conflict
within the 2 nt region were least efficiently joined (Figure 5.1B, left panel, lane 3;
right panel, lane 3 and Figure 5.2, lane 3). Hence, the two DNA ends behave

100
according to a model where they initially anneal and then are subject to steric
constraints on what bases can occupy the 3’ overhangs, and these limitations
affect ligation across any gaps in the top or bottom strands (Figure 5.3).

101
Figure 5.2 Sequence dependence of joining by XRCC4:DNA ligase IV.
(A) DNA substrates with various 3’ overhang sequences were designed
and tested for any sequence dependence. A star indicates the position of the
radioisotope label. The two boxed examples show the sequence at the junction. In
the absence of polymerase mu, no nucleotides are added, and hence, the ligation
must occur across a 1 nt gap. The substrates below the boxed examples simply
show the substrate end sequence.
(B) In each reaction, 20 nM substrate was incubated with 25 nM Ku, 50 nM
X4-LIV, and with or without 100 nM XLF in a 10 uL reaction for 30 min at 37
o
C.
After incubation, reactions were deproteinized and analyzed by 8% denaturing
PAGE. Ligation products were quantified and ligation efficiencies were provided
along with each substrate. XLF stimulation fold was also calculated.

102
Figure 5.2, continued

103
Figure 5.3 Steric hindrance model for terminal sequence effects on ligation
efficiency by XRCC4:DNA ligase IV.
The sequence dependence for the joining of two partially compatible DNA
ends is most simply explained as follows. First, the two 3' overhangs anneal at any
sites of terminal microhomology, which is 2 nts in the cases studied here. Second,
the two DNA ends must orient in a manner that permits the 1 nt gap to be 'closed'
and thereby permit ligation. This step is subject to steric constraints between any
purines (R) at position N on the top strand but at N+1 on the bottom strand. Any
such R:R steric conflicts after the reconfiguration reduce the efficiency of actual
ligation across the gap. In the specific example shown, the G at the 3' end of the
top strand of the left DNA end and the A across from it on the bottom strand of the
right end would represent an R:R conflict as the ligase complex attempts to ligate
across the 1 nt gap.

104
XLF effectively stimulates incompatible or inefficient end ligation by
XRCC:DNA ligase IV, in addition to compatible end joining done at
physiological Mg
2+
concentrations
As mentioned above, we find that ligation of pairs of DNA ends by
XRCC4:DNA ligase IV are highly stimulated for ligation by XLF. However,
compatible DNA ends (4 bp 3’ overhangs) and pairs of blunt DNA ends are not
stimulated for ligation by XLF (Figure 5.4). This suggests that substantial terminal
homology achieves the same effect as XLF for incompatible DNA ends, namely,
stabilization.
Work from our lab and others had shown that XLF can stimulate ligation of
compatible DNA ends (Hentges et al., 2006; Lu et al., 2007). However, those
studies were done at concentrations of Mg
2+
(2.5 mM) that are closer to
physiological free Mg
2+
concentration [~0.3 mM; (Hwang et al., 1993)]. We
reasoned that our failure to detect XLF stimulation for compatible DNA ends here
(Figure 5.4B) might be due to use of a higher Mg
2+
concentration (10 mM) in the
ligation buffer in the current study. To test this, we studied the ligation as a
function of the Mg
2+
concentration. At concentrations of Mg
2+
of 0.2, 0.5, 1 or 2
mM, XLF stimulated ligation of the compatible DNA ends. XLF failed to stimulate
at concentrations ranging from 5 to 20 mM Mg
2+
(Figure 5.5A). For incompatible
DNA end joining, XLF stimulated ligation by XRCC4:DNA ligase IV at all Mg
2+

105
concentrations between 0.2 and 20 mM (Figure 5.5B and Figure 5.6). These
observations help to unify the disparate findings reported by various laboratories
in which some found XLF stimulation of compatible DNA end ligation (Hentges et
al., 2006; Lu et al., 2007) and others did not (Tsai et al., 2007). Like terminal
microhomology, XLF and Mg
2+
may help to stabilize the pair of DNA ends within
the ligase complex. For ends that are already stabilized by terminal
microhomology and high Mg
2+
(5 mM or above), XLF may have no additional
stabilizing effect.

106
Figure 5.4 XLF stimulates incompatible end ligation by XRCC4:DNA ligase
IV but not compatible and blunt end ligations.
(A) Substrates with compatible, incompatible 3’ overhangs and blunt ends
were designed and tested for their ligation efficiency with or without XLF. A star
indicates the position of the radioisotope label. In the ligations, a dimer, e.g.
involves two molecules of the same species joined head to tail.
(B) and (C), In each reaction, 20 nM substrate was incubated with the
protein(s) indicated above the lane in a 10 ul reaction for 30 min at 37
o
C. After
incubation, reactions were deproteinized and analyzed by 8% denaturing PAGE.
Protein concentrations: Ku, 25 nM; X4-LIV, 50 nM; XLF, 100 nM. ‘M’ indicates 50
bp DNA ladder. DNA sequence analysis confirmed that the junctional sequences
conformed to those shown in A.

107
Figure 5.4, continued

108
Figure 5.5 XLF stimulation of ligation as a function of Mg
2+
concentration.
(A) and (B) Compatible substrate JG*163/166 and incompatible substrate
JG*163/186 were tested for the XLF stimulation at various Mg
2+
concentrations. In
each reaction, 20 nM substrate was incubated with 25 nM Ku, 50 nM X4-LIV and
with or without 100 nM XLF in a 10 ul reaction with Mg
2+
concentrations varying
from 0 to 20 mM. For substrate JG*163/166, reactions were carried out at 37
o
C for
2.5 min; for substrate JG*163/186, reactions were carried out at 37
o
C for 30 min.
After incubation, reactions were deproteinized and analyzed by 8% denaturing
PAGE.
(C) Ligation products were quantified and ligation efficiencies were
provided along with each substrate under different Mg
2+
concentrations. The fold
of XLF stimulation was calculated.

109
Figure 5.6 XLF increases the ligation efficiency for incompatible DNA ends
joined by XRCC4:DNA ligase IV.
An incompatible DNA substrate (JG*163/186) was tested for ligation by
XRCC4:DNA ligase IV and Ku with or without XLF stimulation as a function of time.

110
Discussion
Sequence effects on the ligation by XRCC4:DNA ligase IV
Regardless of any flexibility in the association of the DNA ends with the
ligase complex, it is clear that sequence effects are surprisingly large for both
duplex DNA ends and for single-stranded DNA. We do not know the bases for all
aspects of the unusual sequence preferences. Some of these can be accounted
for by the R:R conflict considerations suggested above. However, the poly-dT
preference relative to other sequences for the single-strand DNA end ligation is
unusual. Polymerase mu is known to add nucleotides in a template independent
manner, and it prefers to add pyrimidines, particularly dT (Gu et al., 2007a). It is
possible that DNA ligase IV evolved to have preferred noncovalent bonding sites
for poly-dT overhangs, and this may also apply for single-stranded poly-dT DNA.
In other words, the polymerase mu dT addition preference may have lead to a
selective advantage for a ligase with the dT sequence preference. That is, the
sequence preference of the ligase may have evolved to match that of the
polymerase or vice versa. Though there is no direct evidence that the properties
described in this purified system correspond to in vivo events, there are
pathological and physiological junctional sequences that have remained without
explanation, and which might now be explained by the features reported here.
The propensity of XRCC4:DNA ligase IV to ligate dT overhangs (and the

111
propensity of polymerase mu to add dT and dC) may explain the pyrimidine-rich,
particularly dT, additions at many NHEJ junctions at chromosomal translocations
sites [the translocations from cells that do not express TdT (Roth et al., 1989)]. In
addition, these propensities may explain the pyrimidine-rich, particularly dT-rich,
signal joint additions in pre-B cells that lack TdT expression (Lieber et al., 1988).
These pyrimidine-rich additions have heretofore been perplexing. The evolution of
a polymerase and a ligase with preferences for such sequences may provide an
explanation for them.
The distinctive ability of DNA ligase IV to join incompatible or gapped DNA
ends may depend on its ability to contort the ends into a configuration that can be
joined, or to accept a contorted substrate. Based on our observations, we propose
that the purine/pyrimidine structure of the DNA ends is a key factor. Specifically,
the active site of the ligase may not be sized or structured to accept DNA ends
that place purines near one another on opposing strands, or to contort these ends
sufficiently to ligate them. However, the contortion capability of the ligase and the
flexibility of the DNA ends are two aspects of the same enzyme:substrate
interaction.

112
Degree of XLF stimulation is largest for pairs of DNA ends that are ligated
with the lowest efficiency
At physiological concentrations of Mg
2+
, XLF stimulates the ligation of pairs
of DNA ends by XRCC4:DNA ligase IV, regardless of any stability due to terminal
base pairing. However, at higher Mg
2+
concentrations (>5 mM), terminal base
pairing makes any stabilization by XLF negligible. It is important to note that both
physiological breaks [due to V(D)J recombination or class switch recombination]
or pathologic breaks typically do not have very much terminal base pairing
beyond the chance of 1 or 2 nt. Given the cellular concentration of free Mg
2+
[~0.3
mM; (Hwang et al., 1993)], it is clear that the stimulation provided by XLF is a
major contribution to incompatible DNA end joining. Given that
supra-physiological concentrations of Mg
2+
plus terminal base pairing make XLF
dispensible, there is the possibility that XLF, high Mg
2+
and terminal base pairing
all serve to stabilize the binding of the two DNA ends within the XRCC4:DNA
ligase IV complex.

113
CHAPTER 6
NHEJ BIOCHEMICAL RECONSTITUTION AND ANTI-CANCER TREATMENT
Over the last decade, there have been many trials to reconstitute the NHEJ
pathway, at least partially. In 2004, our lab developed an elegant and
sophisticated NHEJ reconstitution system, which was the first time to reconstitute
the NHEJ pathway in vitro with all the purified components known at that time (Ma
et al., 2004). This study established that polymerase mu and polymerase lambda
are recruited by Ku to the NHEJ pathway via their BRCT domains. Many of the in
vivo findings have been recapitulated in this reconstitution system as well,
including the junction diversity.
However, additional efforts are warranted in the NHEJ reconstitution study.
First, the final ligation in the above reconstitution system is detected by PCR
assay, which may provide some level of artifacts and mask other findings. Thus,
an efficient NHEJ reconstitution system with ligation detection via direct gel needs
to be developed, which would also facilitate the large scale screening process.
Second, in 2006, XLF or Cernunnos has been identified as the new essential
player in NHEJ pathway. Addition of XLF or Cernunnos into the reconstitution
system is therefore necessary.
Here, we presented studies with an efficient partial NHEJ biochemical
reconstitution system. We successfully detected the final NHEJ products with

114
direct gel assay rather than PCR. In the current system, we also established the
functions for many of the NHEJ components. Protein Ku helps XRCC4:DNA
ligase IV stabilize on the DNA ends, thus improving the ligation efficiency. It also
recruits polymerase lambda and polymerase mu to fill in the alignment based
gaps. In addition, polymerase mu is able to perform template independent
addition on the incompatible DNA ends, which provides the terminal
microhomology for ligation. The newly identified XLF can specifically stimulate the
ligation of incompatible and hindered DNA substrates. But its role is fully
dependent on the function of protein Ku, which may imply that Ku also recruits
XLF to the DNA ends (Yano et al., 2008). Surprisingly, XRCC4:DNA ligase IV has
the distinctive and extraordinary capabilities to ligate across gaps and directly
ligate fully incompatible DNA ends in the absence of polymerase and nuclease.
Those novel findings help us further understand the molecular mechanism of
human nonhomologous DNA end joining pathway. Moreover, they may also
provide applications in recombinant DNA technology.
Currently, we are trying to include Artemis and DNA-PKcs into this efficient
reconstitution system. With the efficient NHEJ reconstitution system in hand, we
may have the opportunity to dissect the order of recruitment and release of the
components on the DNA ends and test the synergistic or antagonistic effects for

115
the components in the pathway. Furthermore, this system would also permit us to
screen small molecule inhibitors for NHEJ pathway.
Evidence has accumulated that inactivation or inhibition of the NHEJ
pathway would lead to chemosensitization or radiosensitization of the cancer cells
(Pastwa and Malinowski, 2007). Therefore, NHEJ proteins have become the
potential therapeutic targets to treat cancer. Until now, most of the attention has
been focused on DNA-PK. Inhibitors of DNA-PK kinase activity radiosensitize
cells and inhibit DSB repair, making DNA-PK a possible therapeutic target.
However, in the cells, DNA-PK has functions beyond NHEJ pathway, which
makes DNA-PK inhibitors not as effective as expected. On the other hand, the
feature protein for NHEJ, XRCC4:DNA ligase IV has no other known physiological
role other than its function in NHEJ. It would be interesting to develop specific
small molecule inhibitors for XRCC4:DNA ligase IV, which could be combined with
chemotherapy or radiotherapy to treat cancer patients. In addition, those small
molecule inhibitors may be used in improving the efficiency of gene targeting,
which is dependent on the homologous recombination.

116
BIBLIOGRAPHY

Ahnesorg, P., Smith, P. and Jackson, S.P. (2006) XLF interacts with the
XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell,
124, 301-313.

Bertocci, B., De Smet, A., Berek, C., Weill, J.C. and Reynaud, C.A. (2003)
Immunoglobulin kappa light chain gene rearrangement is impaired in mice
deficient for DNA polymerase mu. Immunity, 19, 203-211.

Bertocci, B., De Smet, A., Weill, J.C. and Reynaud, C.A. (2006) Nonoverlapping
functions of DNA polymerases mu, lambda, and terminal
deoxynucleotidyltransferase during immunoglobulin V(D)J recombination in vivo.
Immunity, 25, 31-41.

Brennan, C.A., Manthey, A.E. and Gumport, R.I. (1983) Using T4 RNA ligase with
DNA substrates. Methods Enzymol, 100, 38-52.

Buck, D., Malivert, L., de Chasseval, R., Barraud, A., Fondaneche, M.C., Sanal,
O., Plebani, A., Stephan, J.L., Hufnagel, M., le Deist, F., Fischer, A., Durandy, A.,
de Villartay, J.P. and Revy, P. (2006) Cernunnos, a novel nonhomologous
end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell,
124, 287-299.

Callebaut, I., Malivert, L., Fischer, A., Mornon, J.P., Revy, P. and de Villartay, J.P.
(2006) Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is
homologous to the yeast nonhomologous end-joining factor Nej1. J Biol Chem,
281, 13857-13860.

Chappell, C., Hanakahi, L.A., Karimi-Busheri, F., Weinfeld, M. and West, S.C.
(2002) Involvement of human polynucleotide kinase in double-strand break repair
by non-homologous end joining. Embo J, 21, 2827-2832.

Chen, L., Trujillo, K., Sung, P. and Tomkinson, A.E. (2000) Interactions of the DNA
ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase.
J Biol Chem, 275, 26196-26205.

Covo, S., Blanco, L. and Livneh, Z. (2004) Lesion bypass by human DNA
polymerase mu reveals a template-dependent, sequence-independent nucleotidyl
transferase activity. J Biol Chem, 279, 859-865.

117
Dai, Y., Kysela, B., Hanakahi, L.A., Manolis, K., Riballo, E., Stumm, M., Harville,
T.O., West, S.C., Oettinger, M.A. and Jeggo, P.A. (2003) Nonhomologous end
joining and V(D)J recombination require an additional factor. Proc Natl Acad Sci U
S A, 100, 2462-2467.

Daley, J.M., Laan, R.L., Suresh, A. and Wilson, T.E. (2005a) DNA joint
dependence of pol X family polymerase action in nonhomologous end joining. J
Biol Chem, 280, 29030-29037.

Daley, J.M., Palmbos, P.L., Wu, D. and Wilson, T.E. (2005b) Nonhomologous end
joining in yeast. Annu Rev Genet, 39, 431-451.

Davis, B.J., Havener, J.M. and Ramsden, D.A. (2008) End-bridging is required for
pol mu to efficiently promote repair of noncomplementary ends by
nonhomologous end joining. Nucleic Acids Res, 36, 3085-3094.

DeFazio, L.G., Stansel, R.M., Griffith, J.D. and Chu, G. (2002) Synapsis of DNA
ends by DNA-dependent protein kinase. Embo J, 21, 3192-3200.

Delarue, M., Boule, J.B., Lescar, J., Expert-Bezancon, N., Jourdan, N., Sukumar,
N., Rougeon, F. and Papanicolaou, C. (2002) Crystal structures of a
template-independent DNA polymerase: murine terminal
deoxynucleotidyltransferase. Embo J, 21, 427-439.

Della, M., Palmbos, P.L., Tseng, H.M., Tonkin, L.M., Daley, J.M., Topper, L.M.,
Pitcher, R.S., Tomkinson, A.E., Wilson, T.E. and Doherty, A.J. (2004)
Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair
machine. Science, 306, 683-685.

Dominguez, O., Ruiz, J.F., Lain de Lera, T., Garcia-Diaz, M., Gonzalez, M.A.,
Kirchhoff, T., Martinez, A.C., Bernad, A. and Blanco, L. (2000) DNA polymerase
mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells.
Embo J, 19, 1731-1742.

Downs, J.A. and Jackson, S.P. (2004) A means to a DNA end: the many roles of
Ku. Nat Rev Mol Cell Biol, 5, 367-378.

Ellenberger, T. and Tomkinson, A.E. (2008) Eukaryotic DNA ligases: structural and
functional insights. Annu Rev Biochem, 77, 313-338.

118
Ferguson, D.O. and Alt, F.W. (2001) DNA double strand break repair and
chromosomal translocation: lessons from animal models. Oncogene, 20,
5572-5579.

Garcia-Diaz, M., Dominguez, O., Lopez-Fernandez, L.A., de Lera, L.T., Saniger,
M.L., Ruiz, J.F., Parraga, M., Garcia-Ortiz, M.J., Kirchhoff, T., del Mazo, J., Bernad,
A. and Blanco, L. (2000) DNA polymerase lambda (Pol lambda), a novel
eukaryotic DNA polymerase with a potential role in meiosis. J Mol Biol, 301,
851-867.

Gilfillan, S., Dierich, A., Lemeur, M., Benoist, C. and Mathis, D. (1993) Mice
lacking TdT: mature animals with an immature lymphocyte repertoire. Science,
261, 1175-1178.

Goffin, C., Bailly, V. and Verly, W.G. (1987) Nicks 3' or 5' to AP sites or to
mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase.
Nucleic Acids Res, 15, 8755-8771.

Gong, C., Bongiorno, P., Martins, A., Stephanou, N.C., Zhu, H., Shuman, S. and
Glickman, M.S. (2005) Mechanism of nonhomologous end-joining in
mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C. Nat
Struct Mol Biol, 12, 304-312.

Goodarzi, A.A., Yu, Y., Riballo, E., Douglas, P., Walker, S.A., Ye, R., Harer, C.,
Marchetti, C., Morrice, N., Jeggo, P.A. and Lees-Miller, S.P. (2006) DNA-PK
autophosphorylation facilitates Artemis endonuclease activity. Embo J, 25,
3880-3889.

Grawunder, U., Wilm, M., Wu, X., Kulesza, P., Wilson, T.E., Mann, M. and Lieber,
M.R. (1997) Activity of DNA ligase IV stimulated by complex formation with
XRCC4 protein in mammalian cells. Nature, 388, 492-495.

Grawunder, U., Zimmer, D. and Leiber, M.R. (1998) DNA ligase IV binds to
XRCC4 via a motif located between rather than within its BRCT domains. Curr
Biol, 8, 873-876.

Gu, J. and Lieber, M.R. (2008) Mechanistic flexibility as a conserved theme
across 3 billion years of nonhomologous DNA end-joining. Genes Dev, 22,
411-415.

119
Gu, J., Lu, H., Tippin, B., Shimazaki, N., Goodman, M.F. and Lieber, M.R. (2007a)
XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across
gaps. Embo J, 26, 1010-1023.

Gu, J., Lu, H., Tsai, A.G., Schwarz, K. and Lieber, M.R. (2007b) Single-stranded
DNA ligation and XLF-stimulated incompatible DNA end ligation by the
XRCC4-DNA ligase IV complex: influence of terminal DNA sequence. Nucleic
Acids Res, 35, 5755-5762.

Hentges, P., Ahnesorg, P., Pitcher, R.S., Bruce, C.K., Kysela, B., Green, A.J.,
Bianchi, J., Wilson, T.E., Jackson, S.P. and Doherty, A.J. (2006) Evolutionary and
functional conservation of the DNA non-homologous end-joining protein,
XLF/Cernunnos. J Biol Chem, 281, 37517-37526.

Hoeijmakers, J.H. (2001) Genome maintenance mechanisms for preventing
cancer. Nature, 411, 366-374.

Hwang, D.L., Yen, C.F. and Nadler, J.L. (1993) Insulin increases intracellular
magnesium transport in human platelets. J Clin Endocrinol Metab, 76, 549-553.

Juarez, R., Ruiz, J.F., Nick McElhinny, S.A., Ramsden, D. and Blanco, L. (2006) A
specific loop in human DNA polymerase mu allows switching between creative
and DNA-instructed synthesis. Nucleic Acids Res, 34, 4572-4582.

Kienker, L.J., Shin, E.K. and Meek, K. (2000) Both V(D)J recombination and
radioresistance require DNA-PK kinase activity, though minimal levels suffice for
V(D)J recombination. Nucleic Acids Res, 28, 2752-2761.

Koch, C.A., Agyei, R., Galicia, S., Metalnikov, P., O'Donnell, P., Starostine, A.,
Weinfeld, M. and Durocher, D. (2004) Xrcc4 physically links DNA end processing
by polynucleotide kinase to DNA ligation by DNA ligase IV. Embo J, 23,
3874-3885.

Komori, T., Okada, A., Stewart, V. and Alt, F.W. (1993) Lack of N regions in
antigen receptor variable region genes of TdT-deficient lymphocytes. Science,
261, 1171-1175.

Kuhn, H. and Frank-Kamenetskii, M.D. (2005) Template-independent ligation of
single-stranded DNA by T4 DNA ligase. Febs J, 272, 5991-6000.

120
Kurimasa, A., Kumano, S., Boubnov, N.V., Story, M.D., Tung, C.S., Peterson, S.R.
and Chen, D.J. (1999) Requirement for the kinase activity of human
DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.
Mol Cell Biol, 19, 3877-3884.

Lee, J.W., Blanco, L., Zhou, T., Garcia-Diaz, M., Bebenek, K., Kunkel, T.A., Wang,
Z. and Povirk, L.F. (2004) Implication of DNA polymerase lambda in
alignment-based gap filling for nonhomologous DNA end joining in human nuclear
extracts. J Biol Chem, 279, 805-811.

Lieber, M.R. (2008) The mechanism of human nonhomologous DNA end joining. J
Biol Chem, 283, 1-5.

Lieber, M.R., Hesse, J.E., Mizuuchi, K. and Gellert, M. (1988) Lymphoid V(D)J
recombination: nucleotide insertion at signal joints as well as coding joints. Proc
Natl Acad Sci U S A, 85, 8588-8592.

Lieber, M.R., Lu, H., Gu, J. and Schwarz, K. (2008) Flexibility in the order of action
and in the enzymology of the nuclease, polymerases, and ligase of vertebrate
non-homologous DNA end joining: relevance to cancer, aging, and the immune
system. Cell Res, 18, 125-133.

Lieber, M.R., Ma, Y., Pannicke, U. and Schwarz, K. (2003) Mechanism and
regulation of human non-homologous DNA end-joining. Nat Rev Mol Cell Biol, 4,
712-720.

Lu, H., Pannicke, U., Schwarz, K. and Lieber, M.R. (2007) Length-dependent
binding of human XLF to DNA and stimulation of XRCC4.DNA ligase IV activity. J
Biol Chem, 282, 11155-11162.

Ma, Y ., Lu, H., Schwarz, K. and Lieber, M.R. (2005a) Repair of double-strand DNA
breaks by the human nonhomologous DNA end joining pathway: the iterative
processing model. Cell Cycle, 4, 1193-1200.

Ma, Y., Lu, H., Tippin, B., Goodman, M.F., Shimazaki, N., Koiwai, O., Hsieh, C.L.,
Schwarz, K. and Lieber, M.R. (2004) A biochemically defined system for
mammalian nonhomologous DNA end joining. Mol Cell, 16, 701-713.

121
Ma, Y., Pannicke, U., Schwarz, K. and Lieber, M.R. (2002) Hairpin opening and
overhang processing by an Artemis/DNA-dependent protein kinase complex in
nonhomologous end joining and V(D)J recombination. Cell, 108, 781-794.

Ma, Y., Schwarz, K. and Lieber, M.R. (2005b) The Artemis:DNA-PKcs
endonuclease cleaves DNA loops, flaps, and gaps. DNA Repair (Amst), 4,
845-851.

Mahajan, K.N., Gangi-Peterson, L., Sorscher, D.H., Wang, J., Gathy, K.N.,
Mahajan, N.P., Reeves, W.H. and Mitchell, B.S. (1999) Association of terminal
deoxynucleotidyl transferase with Ku. Proc Natl Acad Sci U S A, 96,
13926-13931.

Mahajan, K.N., Nick McElhinny, S.A., Mitchell, B.S. and Ramsden, D.A. (2002)
Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu
in end-joining double-strand break repair. Mol Cell Biol, 22, 5194-5202.

Mahaney, B.L., Meek, K. and Lees-Miller, S.P. (2009) Repair of ionizing
radiation-induced DNA double-strand breaks by non-homologous end-joining.
Biochem J, 417, 639-650.

Meek, K., Dang, V. and Lees-Miller, S.P. (2008) DNA-PK: the means to justify the
ends? Adv Immunol, 99, 33-58.

Moon, A.F., Garcia-Diaz, M., Bebenek, K., Davis, B.J., Zhong, X., Ramsden, D.A.,
Kunkel, T.A. and Pedersen, L.C. (2007) Structural insight into the substrate
specificity of DNA Polymerase mu. Nat Struct Mol Biol, 14, 45-53.

Moshous, D., Callebaut, I., de Chasseval, R., Corneo, B., Cavazzana-Calvo, M.,
Le Deist, F., Tezcan, I., Sanal, O., Bertrand, Y., Philippe, N., Fischer, A. and de
Villartay, J.P. (2001) Artemis, a novel DNA double-strand break repair/V(D)J
recombination protein, is mutated in human severe combined immune deficiency.
Cell, 105, 177-186.

Nick McElhinny, S.A., Havener, J.M., Garcia-Diaz, M., Juarez, R., Bebenek, K.,
Kee, B.L., Blanco, L., Kunkel, T.A. and Ramsden, D.A. (2005) A gradient of
template dependence defines distinct biological roles for family X polymerases in
nonhomologous end joining. Mol Cell, 19, 357-366.

122
Nick McElhinny, S.A. and Ramsden, D.A. (2003) Polymerase mu is a
DNA-directed DNA/RNA polymerase. Mol Cell Biol, 23, 2309-2315.

Nick McElhinny, S.A., Snowden, C.M., McCarville, J. and Ramsden, D.A. (2000)
Ku recruits the XRCC4-ligase IV complex to DNA ends. Mol Cell Biol, 20,
2996-3003.

Niewolik, D., Pannicke, U., Lu, H., Ma, Y., Wang, L.C., Kulesza, P., Zandi, E.,
Lieber, M.R. and Schwarz, K. (2006) DNA-PKcs dependence of Artemis
endonucleolytic activity, differences between hairpins and 5' or 3' overhangs. J
Biol Chem, 281, 33900-33909.

O'Driscoll, M., Cerosaletti, K.M., Girard, P.M., Dai, Y., Stumm, M., Kysela, B.,
Hirsch, B., Gennery, A., Palmer, S.E., Seidel, J., Gatti, R.A., Varon, R., Oettinger,
M.A., Neitzel, H., Jeggo, P.A. and Concannon, P. (2001) DNA ligase IV mutations
identified in patients exhibiting developmental delay and immunodeficiency. Mol
Cell, 8, 1175-1185.

Pastwa, E. and Malinowski, M. (2007) Non-homologous DNA end joining in
anticancer therapy. Curr Cancer Drug Targets, 7, 243-250.

Ramadan, K., Maga, G., Shevelev, I.V., Villani, G., Blanco, L. and Hubscher, U.
(2003) Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl
transferase activity and can elongate RNA primers: implications for novel
functions. J Mol Biol, 328, 63-72.

Ramadan, K., Shevelev, I.V., Maga, G. and Hubscher, U. (2004) De novo DNA
synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal
deoxyribonucleotidyl transferase. J Mol Biol, 339, 395-404.

Ramsden, D.A. and Gellert, M. (1998) Ku protein stimulates DNA end joining by
mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand
breaks. Embo J, 17, 609-614.

Riballo, E., Critchlow, S.E., Teo, S.H., Doherty, A.J., Priestley, A., Broughton, B.,
Kysela, B., Beamish, H., Plowman, N., Arlett, C.F., Lehmann, A.R., Jackson, S.P.
and Jeggo, P.A. (1999) Identification of a defect in DNA ligase IV in a
radiosensitive leukaemia patient. Curr Biol, 9, 699-702.

123
Riballo, E., Kuhne, M., Rief, N., Doherty, A., Smith, G.C., Recio, M.J., Reis, C.,
Dahm, K., Fricke, A., Krempler, A., Parker, A.R., Jackson, S.P., Gennery, A.,
Jeggo, P.A. and Lobrich, M. (2004) A pathway of double-strand break rejoining
dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci. Mol
Cell, 16, 715-724.

Rich, T., Allen, R.L. and Wyllie, A.H. (2000) Defying death after DNA damage.
Nature, 407, 777-783.

Robins, P. and Lindahl, T. (1996) DNA ligase IV from HeLa cell nuclei. J Biol
Chem, 271, 24257-24261.

Roth, D.B., Chang, X.B. and Wilson, J.H. (1989) Comparison of filler DNA at
immune, nonimmune, and oncogenic rearrangements suggests multiple
mechanisms of formation. Mol Cell Biol, 9, 3049-3057.

Rothkamm, K., Kruger, I., Thompson, L.H. and Lobrich, M. (2003) Pathways of
DNA double-strand break repair during the mammalian cell cycle. Mol Cell Biol,
23, 5706-5715.

Schar, P., Herrmann, G., Daly, G. and Lindahl, T. (1997) A newly identified DNA
ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of
DNA double-strand breaks. Genes Dev, 11, 1912-1924.

Schlissel, M.S. (1998) Structure of nonhairpin coding-end DNA breaks in cells
undergoing V(D)J recombination. Mol Cell Biol, 18, 2029-2037.

Shimazaki, N., Yoshida, K., Kobayashi, T., Toji, S., Tamai, K. and Koiwai, O. (2002)
Over-expression of human DNA polymerase lambda in E. coli and
characterization of the recombinant enzyme. Genes Cells, 7, 639-651.

Snopek, T.J., Sugino, A., Agarwal, K.L. and Cozzarelli, N.R. (1976) Catalysis of
DNA joining by bacteriophage T4 RNA ligase. Biochem Biophys Res Commun, 68,
417-424.

Sonoda, E., Hochegger, H., Saberi, A., Taniguchi, Y. and Takeda, S. (2006)
Differential usage of non-homologous end-joining and homologous recombination
in double strand break repair. DNA Repair (Amst), 5, 1021-1029.

124
Spagnolo, L., Rivera-Calzada, A., Pearl, L.H. and Llorca, O. (2006)
Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex
assembled on DNA and its implications for DNA DSB repair. Mol Cell, 22,
511-519.

Suwa, A., Hirakata, M., Takeda, Y ., Jesch, S.A., Mimori, T. and Hardin, J.A. (1994)
DNA-dependent protein kinase (Ku protein-p350 complex) assembles on
double-stranded DNA. Proc Natl Acad Sci U S A, 91, 6904-6908.

Teo, S.H. and Jackson, S.P. (1997) Identification of Saccharomyces cerevisiae
DNA ligase IV: involvement in DNA double-strand break repair. Embo J, 16,
4788-4795.

Tippin, B., Kobayashi, S., Bertram, J.G. and Goodman, M.F. (2004) To slip or skip,
visualizing frameshift mutation dynamics for error-prone DNA polymerases. J Biol
Chem, 279, 45360-45368.

Tomkinson, A.E., Vijayakumar, S., Pascal, J.M. and Ellenberger, T. (2006) DNA
ligases: structure, reaction mechanism, and function. Chem Rev, 106, 687-699.

Tsai, C.J., Kim, S.A. and Chu, G. (2007) Cernunnos/XLF promotes the ligation of
mismatched and noncohesive DNA ends. Proc Natl Acad Sci U S A, 104,
7851-7856.

Tseng, H.M. and Tomkinson, A.E. (2002) A physical and functional interaction
between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in
nonhomologous end joining. J Biol Chem, 277, 45630-45637.

van Heemst, D., den Reijer, P.M. and Westendorp, R.G. (2007) Ageing or cancer:
a review on the role of caretakers and gatekeepers. Eur J Cancer, 43, 2144-2152.

Walker, J.R., Corpina, R.A. and Goldberg, J. (2001) Structure of the Ku
heterodimer bound to DNA and its implications for double-strand break repair.
Nature, 412, 607-614.

Wilson, T.E., Grawunder, U. and Lieber, M.R. (1997) Yeast DNA ligase IV
mediates non-homologous DNA end joining. Nature, 388, 495-498.

125
Wilson, T.E. and Lieber, M.R. (1999) Efficient processing of DNA ends during
yeast nonhomologous end joining. Evidence for a DNA polymerase beta
(Pol4)-dependent pathway. J Biol Chem, 274, 23599-23609.

Wu, P.Y., Frit, P., Meesala, S., Dauvillier, S., Modesti, M., Andres, S.N., Huang, Y.,
Sekiguchi, J., Calsou, P., Salles, B. and Junop, M.S. (2009) Structural and
functional interaction between the human DNA repair proteins DNA ligase IV and
XRCC4. Mol Cell Biol, 29, 3163-3172.

Yano, K., Morotomi-Yano, K., Wang, S.Y., Uematsu, N., Lee, K.J., Asaithamby, A.,
Weterings, E. and Chen, D.J. (2008) Ku recruits XLF to DNA double-strand
breaks. EMBO Rep, 9, 91-96.

Yoo, S. and Dynan, W.S. (1999) Geometry of a complex formed by double strand
break repair proteins at a single DNA end: recruitment of DNA-PKcs induces
inward translocation of Ku protein. Nucleic Acids Res, 27, 4679-4686.

Yu, K. and Lieber, M.R. (2000) The nicking step in V(D)J recombination is
independent of synapsis: implications for the immune repertoire. Mol Cell Biol, 20,
7914-7921.

Abstract (if available)

Abstract DNA double-strand breaks (DSBs) represent the most deleterious form of DNA damage, as both of the DNA strands are broken. In mammalian cells, DSBs are repaired predominantly by nonhomologous DNA end joining (NHEJ) pathway. NHEJ functions throughout the cell cycle to repair such lesions. Defects in NHEJ result in marked sensitivity to ionizing radiation and ablation of lymphocytes, which rely on NHEJ to complete the rearrangement of antigen-receptor genes. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes, but which might also contribute to some of the genetic changes that underlie cancer and aging. To further understand the mechanism of human nonhomologous DNA end joining pathway, we performed in vitro biochemical reconstitution assay. Here, we present some distinctive features of polymerase mu and the ligase complex, XRCC4:DNA ligase IV:XLF.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Molecular elucidation of nonhomologous DNA end-joining in the context of nucleosome core particles

PDF

Studies on the role of Artemis in non-homologous DNA end-joining to understand the mechanism and discover therapies

PDF

Mechanisms of nucleases in non-homologous DNA end joining

PDF

Current models of non-homologous end joining and their implications in gene therapy

PDF

X-ray structural studies on DNA-dependent protein kinase catalytic subunit:DNA co-crystals

PDF

Biochemistry and reconstitution of V(D)J recombination in a purified system

PDF

The mechanism of R-loop formation in mammalian immunoglobulin class switch recombination

PDF

Unraveling the molecular mechanisms of heterochromatic double strand break repair in Drosophila cells

PDF

Mechanistic basis for chromosomal translocations at the E2A gene

PDF

Nutritional competence in Escherichia coli: the use of double-stranded DNA as a nutrient

PDF

KAT5 acetyltransferase in DNA damage repair in Schizosaccharomyces pombe

PDF

C. elegans topoisomerase II regulates chromatin architecture and DNA damage for germline genome activation

PDF

Molecular mechanisms of recurrent chromosomal translocations in human leukemias and lymphomas

PDF

Decoding protein-DNA binding determinants mediated through DNA shape readout

PDF

New tools for whole-genome analysis of DNA replication timing and fork elongation in saccharomyces cerevisiae

PDF

The mechanism of mammalian immunoglobulin class switch recombination: R-loop structures and activation-induced deaminase site preferences

PDF

The mechanism of mammalian immunoglobulin class switch recombination

PDF

Characterizing and manipulating homology-directed gene editing in human cells

PDF

Subnuclear localization of replication origins is controlled by Fkh1-dependent recruitment of DDK to origins in S. cerevisiae

PDF

Mechanism study of SV40 large tumor antigen atpase and helicase functions in viral DNA replication

Asset Metadata

Creator Gu, Jiafeng (author)

Core Title Mechanism of human nonhomologous DNA end joining

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 07/15/2009

Defense Date 06/01/2009

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

Tag biochemical reconstitution,cancer treatment,DNA double strand break,DNA repair and recombination,nonhomologous DNA end joining,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lieber, Michael R. (committee chair), Aparicio, Oscar Martin (committee member), Chen, Xiaojiang (committee member), Goodman, Steven (committee member), Zandi, Ebrahim (committee member)

Creator Email jiafengg@usc.edu,porter_gu@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2366

Unique identifier UC1138194

Identifier etd-Gu-3003 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-407480 (legacy record id),usctheses-m2366 (legacy record id)

Legacy Identifier etd-Gu-3003.pdf

Dmrecord 407480

Document Type Dissertation

Rights Gu, Jiafeng

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu