University of Southern California Dissertations and Theses

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

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

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MOLECULAR ELUCIDATION OF NONHOMOLOGOUS DNA
END-JOINING IN THE CONTEXT OF NUCLEOSOME CORE PARTICLES

by

Christina Anastasia Gerodimos

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

August 2021

Copyright 2021 Christina Anastasia Gerodimos
ii

ACKNOWLEDGEMENTS
Firstly, I’d like to thank my advisor Dr. Michael Lieber. As a foremost expert in the field
of DNA repair, his insight and direct engagement in the conceptualization, design, and execution
of these studies were invaluable. With Dr. Lieber’s mentorship I’ve developed skills that I’ll
carry throughout the course of my life, in and out of the lab.
I’d like to thank my committee members Dr. Ebrahim Zandi and Dr. Woojin An for their
advice and guidance as I progressed through my program. I’d also like to thank Dr. Chih-Lin
Hsieh, whose vast knowledge and expertise was vital when I needed to optimize protocols or
troubleshoot experiments.
Additionally, I’d like to thank former and current members of the Lieber and Hsieh labs
for their kindness, encouragement, and constant willingness to help: Dr. Howard Chang, Dr.
Nicholas Pannunzio, Dr. Shuangchao Ma, Dr. Anne Esguerra, Dr. Go Watanabe, Cindy Okitsu,
Dr. Bailin Zhao, and Di Liu.
Finally, I’d like to thank my parents, Angelo and Soyon Gerodimos, who have been with
me every step of the way. I can’t express how very much I appreciate their support over the last
six years, and the 22 before that—thank you, I love you, and I couldn’t have done it without you.

iii

TABLE OF CONTENTS

Acknowledgements ii

List of Figures v

Abbreviations vi

Abstract vii

Chapter 1 OVERVIEW 1
1. Significance 1
2. Causes and consequences of DNA double-strand breaks 1
3. General steps of the NHEJ pathway 2
4. The Artemis:DNA-PKcs nuclease complex 4
5. DNA polymerases: Pol μ, Pol λ, and TdT 5
6. The XLF:XRCC4:DNA ligase IV complex 5
7. NHEJ in the context of chromatin 6
8. Clinical significance 7
9. Key questions at the inception of this thesis project 7

Chapter 2 EXPERIMENTAL PROCEDURES 11
1. Oligonucleotides and DNA substrates 11
2. Protein expression and purification 15
3. Nuclease assay 16
4. Ligation assay 17
5. NHEJ assay 17
6. Junction sequence analysis 17
7. Nucleosome reconstitution assay 18
8. Mononucleosome NHEJ assay 18
9. Dinucleosome ligation assay 19

Chapter 3 EFFECTS OF DNA END CONFIGURATION ON XRCC4:DNA
LIGASE IV AND ITS STIMULATION OF ARTEMIS ACTIVITY 21
Abstract 21
Introduction 22
Results 23
Discussion 37

Chapter 4 POLYMERASE ACTIVITY OF CHIMERIC TERMINAL
DEOXYNUCLEOTIDYLTRANSFERASE 43
Abstract 43
Results 43
Discussion 46

iv

Chapter 5 NONHOMOLOGOUS DNA END JOINING OF NUCLEOSOMAL
SUBSTRATES IN A PURIFIED SYSTEM 49
Abstract 49
Introduction 49
Results 50
Discussion 59

Concluding Remarks 64

Bibliography 67

v

LIST OF FIGURES

Figure 1.1 4

Figure 1.2 8

Figure 2.1 14

Figure 3.1 25

Figure 3.2 26

Figure 3.3 28

Figure 3.4 29

Figure 3.5 30

Figure 3.6 32

Figure 3.7 34

Figure 3.8 36

Figure 3.9 38

Figure 4.1 45

Figure 5.1 51

Figure 5.2 52

Figure 5.3 54

Figure 5.4 55

Figure 5.5 56

Figure 5.6 58

Figure 5.7 59

Figure 5.8 60

Figure 5.9 61
vi

ABBREVIATIONS
DSB – double-strand break
NHEJ – nonhomologous DNA end-joining
HR – homologous recombination
HDR – homology-directed repair
IR – ionizing radiation
ROS – reactive oxygen species
RAG – recombination activating gene
AID – activation-induced deaminase
DNA-PKcs – DNA-dependent protein kinase, catalytic subunit
XRCC4 – X-ray repair cross-complementing protein 4
XLF – XRCC4-like factor
PALF – polynucleotide kinase and aprataxin-like forkhead-associated protein
SCID – severe combined immunodeficiency
Pol μ – polymerase μ
Pol λ – polymerase λ
TdT – terminal deoxynucleotidyl transferase
BRCT – breast cancer type 1 susceptibility protein C-terminal
NCP – nucleosome core particle
bp – base pair(s)
SSBR – single-strand base repair
X4:LIV – XRCC4:DNA ligase IV
OH – hydroxyl group
nt – nucleotide(s)
PAXX – paralog of XRCC4 and XLF

vii

Abstract
DNA double-strand breaks (DSBs) are a common occurrence in eukaryotic cells and are
potentially the most seriously damaging to the genome because, if unrepaired, they can result in
the loss of entire arms of chromosomes, containing thousands of genes. Nonhomologous DNA
end-joining (NHEJ) and homologous recombination are major pathways for repair of these breaks.
In vertebrates, all DSBs are pathologic except those generated during V(D)J recombination and
class switch recombination. Pathologic breaks arise from multiple sources, including ionizing
radiation, reactive oxygen species, and aberrant action by nucleases involved in normal cellular
processes. NHEJ, the predominant pathway for repair of both physiologic and pathologic DSBs,
involves several core components which are used to ligate a wide variety of DNA ends that differ
in chemistry, sequence, and structure. The NHEJ core components have evolved to converge upon
a theme of mechanistic flexibility which allows for the processing of such a variety of substrates.
As in most DNA repair pathways, these components include a nuclease, polymerases, and a ligase
complex, all of which, in addition to provisional accessory factors, are utilized for repair. Though
the proteins of the NHEJ pathway have been genetically identified, a precise mechanistic
understanding of this process has yet to be attained. For a better functional understanding, the
components of NHEJ must be biochemically characterized. As detailed in this dissertation, I
employed biochemical methods to study the NHEJ pathway using free DNA as well as
nucleosomal substrates to gain an improved understanding of how NHEJ occurs at various DNA
end configurations. More specifically, I tested the ability of the NHEJ ligase complex to stimulate
the rate of NHEJ-dependent joining of different substrates. Additionally, I assessed the role of a
5’ phosphate in improving NHEJ efficiency. Finally, I used a biochemical NHEJ reconstitution
system to determine how NHEJ is carried out in the context of chromatin. The insights gained
viii

from these studies contribute to our current functional model of NHEJ and may prove crucial in
the development of DNA repair-targeted cancer therapies.

1

CHAPTER 1.
OVERVIEW

1.1 SIGNIFICANCE
DNA DSBs are common events, with an estimate of 10 DSBs per day per cell based on dividing
early passage primary human and mouse fibroblasts (Martin et al., 1985; Lieber & Karanjawala,
2004). If unrepaired, DSBs are potentially the most damaging type of genetic lesion, resulting in
cell death due to the loss of entire chromosome arms or by induction of p53-mediated apoptosis
(Rich et al., 2000). Two major pathways are used for repairing these breaks: homologous
recombination (HR) and NHEJ. In diploid organisms and replicating bacteria and yeast, the
presence of a homology donor allows for homology-directed repair (HDR) to be carried out
through HR or, less commonly, single-strand annealing and breakage-induced replication. The
absence of a nearby homology donor necessitates another form of DSB repair, making NHEJ the
preferred pathway outside of late S and G2 phases (Renkawitz et al., 2014; Symington & Gautier,
2011). Early in evolution, the availability of another form of DSB repair conferred a survival
advantage, which is reflected in the mechanistic flexibility shared by key factors of NHEJ (Gu &
Lieber, 2008).

1.2 CAUSES AND CONSEQUENCES OF DNA DOUBLE-STRAND BREAKS
In vertebrates, all DSBs are pathologic except for those created during the early stages of
lymphocyte development. Pathologic DSBs arise from multiple sources including ionizing
radiation (IR), inadvertent action by nuclear enzymes, and reactive oxygen species (ROS)
generated during oxidative metabolism (Lieber et al., 2003). Sources of ambient IR—primarily,
2

cosmic rays and the radioactive decay of terrestrial metals—contribute to the formation of DSBs
by producing free radicals which cause single-strand breaks in DNA, every 25 of which generates
one DSB in the genome (Lieber, 2010; Santivasi & Xia, 2014). ROS, a normal byproduct of
oxidative metabolism, are produced at a rate of 10
9
per cell per hour and cause DSBs through the
formation of closely spaced single-strand breaks (Chance et al., 1979; Karanjawala et al., 2002).
Inadvertent action by nuclear enzymes can cause DSBs during DNA replication and other major
cellular processes. Type II topoisomerases create transient breaks in the DNA to relieve stress
caused by supercoiling; failure to re-join DNA ends results in the persistence of these breaks
(Ashour et al., 2015). In the nuclei of lymphoid cells, off-target action by enzymes such as the
recombination activating gene (RAG) complex and activation-induced deaminase (AID) accounts
for ~50% of lymphoma-associated chromosomal translocations (Lieber, 2010; Mahowald et al.,
2009). Physiologic breaks in vertebrates are only formed in the early stages of B- and T-cell
development during V(D)J recombination and class switch recombination, which results in the
diversity of T-cell receptors and immunoglobulin isotypes necessary for normal lymphocyte
function. These processes, both of which require the formation of DSBs for recombination to
occur, call for resolution of these breaks by NHEJ (Lu et al., 2007; Matthews & Simmons, 2014).

1.3 GENERAL STEPS OF THE NHEJ PATHWAY
As is the case for most DNA repair pathways, core components of NHEJ include a nuclease,
polymerases, and a ligase. In multicellular eukaryotes, Ku is the first NHEJ factor to be recruited
to the site of a DSB. Ku, a dimer of Ku70 and Ku86, is the most abundant DNA binding protein
in the cell with a strong equilibrium dissociation constant (~10
-10
M) for double-stranded DNA
ends (Mimori & Hardin, 1986; Blier et al., 1993; West et al., 1998). It is likely, then, that Ku serves
3

as a tool belt protein by recognizing DSBs in a sequence-independent manner and recruiting other
factors to the break site (Griffith et al., 1992). After loading onto DNA, Ku undergoes a
conformational change which allows it to interact with other factors, as evidenced by the inability
of Ku to form stable complexes with DNA-dependent protein kinase, catalytic subunit (DNA-
PKcs) in the absence of DNA ends (Yaneva et al., 1997). Depending upon which steps are
necessary for the DNA ends to be ligated, the nuclease, ligase, or polymerases are recruited in any
order, and sometimes iteratively (Ma et al., 2004) (Figure 1.1). In a scenario requiring resection of
the DNA ends, autophosphorylated DNA-PKcs forms a complex with Artemis which is recruited
to the DSB through interaction with Ku (Ma et al., 2005). Upon phosphorylation, Artemis
nucleolytically resects DNA ends at boundaries of single- and double-strandedness. If necessary,
polymerases μ or λ add nucleotides until a region of microhomology is formed, allowing the ends
to anneal and stabilize. Finally, X-ray repair cross-complementing protein 4 (XRCC4), and in
some instances, XRCC4-like factor (XLF)/Cernunnos, act in complex with DNA ligase IV to ligate
the ends (Lieber, 2010). Though there are a considerable number of DNA end conformations that
can arise from a DSB, the mechanistic flexibility exhibited by the NHEJ pathway allows for the
repair of a wide variety of substrates. It follows, then, that NHEJ-mediated joining of these
substrates results in a variety of ligated products. Indeed, though the same starting ends may
undergo repair along a preferred “path,” many products can be generated from a single substrate
(Gu & Lieber, 2008; Chang et al., 2016).
4

Figure 1.1. Overview of the NHEJ mechanism. DSBs are generated throughout the duplex by either physiologic or pathologic
sources. Upon induction of a break, Ku binds to the DSB termini. A conformational change in Ku allows for more efficient
recognition and binding of downstream NHEJ proteins (polymerases, the nuclease complex, or the ligase complex) to the DNA
ends. Iterative processing by NHEJ proteins results in diverse sequences at the repaired junction.

1.4 THE ARTEMIS:DNA-PKcs NUCLEASE COMPLEX
Artemis, also called SNM1C, is a member of the metallo-β-lactamase superfamily of
nucleases. In physiological conditions, the Artemis:DNA-PKcs complex has 5’ and 3’
endonuclease activity as well as hairpin opening activity (Ma et al., 2002; Niewolik et al., 2006;
Gu et al., 2010), whereas Artemis alone can act as a 5’ exonuclease on ssDNA (Li et al., 2014).
Artemis may also act on other DNA substrates (pseudo-Y, 5’ flaps, and symmetrical bubble
structures) (Ma et al., 2005; Chang & Lieber, 2016). Though Artemis, whether alone or in complex
with DNA-PKcs, is a sufficient nuclease for NHEJ, other nucleases such as polynucleotide kinase
and aprataxin-like forkhead-associated protein (PALF, or APLF) are circumstantially involved (Li
5

et al., 2011). Patients deficient in Artemis have severe combined immunodeficiency (SCID), the
defining feature of which is a poor immune response caused by defects in lymphocyte function. A
specific form of SCID has been shown to be associated with a nonsense mutation at amino acid
192 of Artemis (Harrington et al., 1992; Karanjawala et al., 2002; Ege et al., 2005).

1.5 DNA POLYMERASES: POL μ, POL λ, AND TdT
Of the four polymerases comprising the Pol X family, three are involved in NHEJ—
polymerase μ (Pol μ), polymerase λ (Pol λ), and terminal deoxynucleotidyl transferase (TdT).
Polymerase β, which lacks the breast cancer type 1 susceptibility protein C-terminal (BRCT)
domain shared by the other Pol X family members, has a role in base excision repair (Yamtich &
Sweasy, 2010). Unlike polymerases μ and λ, TdT is only expressed in early lymphocytes, limiting
its function to processing of DSBs generated during antigen receptor assembly. TdT is the only
polymerase of the three which can add nucleotides in a completely template-independent manner,
thereby contributing to the genetic diversity of immunoglobulins and T-cell receptors (Komori et
al., 1993; McElhinny et al., 2005). Pol μ has both template-dependent and template-independent
activity, reflecting the mechanistic flexibility exhibited by the NHEJ pathway (Moon et al., 2007;
Davis et al., 2008). While Pol λ is largely template-dependent, it has a lyase domain that is
nonfunctional in Pol μ and TdT which allows it to remove damaged bases (Ramadan et al., 2003).

1.6 THE XLF:XRCC4:DNA LIGASE IV COMPLEX
The ligase complex is essential for NHEJ-mediated joining to occur (Grawunder et al., 1998).
The complex consists of XRCC4 and DNA ligase IV, though DNA ligase IV is capable of ligating
nicks and compatible overhangs in the absence of XRCC4. In complex with XRCC4, DNA ligase
6

IV can ligate substrates with shorter regions of microhomology over 1-nt gaps (Gu et al., 2007).
With Ku, the complex can ligate incompatible ends; efficiency of ligation increases in the presence
of XLF, which interacts with the ligase complex via a head-to-head contact with XRCC4 (Lu et
al., 2007; Gu et al., 2007). Patients missing XLF are radiosensitive and show defects in V(D)J
recombination (Grawunder et al., 1998).

1.7 NHEJ IN THE CONTEXT OF CHROMATIN
In cells, DNA is packaged into chromatin, the fundamental repeating unit of which is the
nucleosome core particle (NCP). The canonical NCP is a nucleoprotein complex comprised of
147 base pairs (bp) of DNA wrapped around a histone octamer containing two H2A/H2B
duplexes and one H3/H4 tetramer (Reid et al., 2017). It has been shown that organization of
DNA into higher order structures necessitates large-scale rearrangement so that damage sites are
made accessible to repair machinery (Smerdon & Lieberman, 1978); however it remains unclear
how repair factors work locally at DSBs in nucleosomal DNA. For in vitro analysis, one early
study used heterogeneous and undefined histones on a DNA substrate that permitted random
positioning of histone octamers (Kysela et al., 2005). Studies using homogeneous histones on
defined nucleosomal substrates have been lacking.
Previous studies have described the importance of several factors influencing nucleosome
accessibility to both single-strand and double-strand DNA repair (Adkins et al., 2013; G. Li &
Widom, 2004; Chafin et al., 2000; Rodriguez & Smerdon, 2013; Hinz et al., 2015; Bilotti et al.,
2017; Fei et al., 2018). The steric challenge presented by the interaction between DNA and the
histone octamer necessitates that the lesion is either already accessible by way of DNA positioning
or made accessible through remodeling of the nucleoprotein complex. In single-strand base repair
7

(SSBR), damaged bases which are oriented inward toward the histone octamer, rather than away
from the octamer surface, are less accessible to recognition by single-strand repair proteins (Hinz
et al., 2015; Rodriguez & Smerdon, 2013). Damaged bases positioned farther away from the dyad
center of the core particle were processed at a slower rate than those near the dyad (Chafin et al.,
2000; Rodriguez & Smerdon, 2013). For double-strand recombination and repair by HR proteins,
exposure of 3’ overhangs and duplex regions of hundreds of base pairs are needed to permit strand
invasion and strand exchange steps intrinsic to HR.

1.8 CLINICAL SIGNIFICANCE
If unrepaired, DSBs can lead to cell death due to the loss of entire chromosome arms. NHEJ is
the predominant pathway by which these breaks are repaired. The objective of this study is to
biochemically characterize the NHEJ pathway. The key components of the NHEJ pathway have
been identified, but there is limited knowledge regarding the mechanisms of these components in
relation to each other. By studying how the order and timing of NHEJ is mediated by DNA end
structure, I hope to contribute to the understanding of how both pathologic and physiologic DSBs
are processed and repaired. In addition to offering a more detailed view of a critical process, such
insights will be integral to the development of therapies which target DNA repair mechanisms in
cancer.

1.9 KEY QUESTIONS AT THE INCEPTION OF THIS THESIS PROJECT
Does X4:LIV stimulate Artemis resection of 3’ overhangs, 5’ overhangs, and blunt ends?
NHEJ reconstitution studies from our lab suggest that Artemis is stimulated by XRCC4:DNA
ligase IV (X4:LIV) in the absence of DNA-PKcs, an activity not previously found to be exhibited
8

by Artemis To explore this, we performed Artemis resection assays using a gel-based biochemical
system which allows us to determine the amount of nucleolytic activity performed by Artemis on
selected duplex DNA substrates. We found that Artemis activity on a substrate with a 3’ 10-nt
overhang is stimulated in the presence of X4:LIV without DNA-PKcs (Figure 1.2, lane 4).
Stimulation of Artemis by X4:LIV without DNA-PKcs has previously not been reported. Testing
DNA substrates with multiple end configurations will potentially reveal another mechanism by
which Artemis processes ends at DSBs.

Figure 1.2. Artemis resection of 3′ overhangs is stimulated by X4·LIV. NHEJ proteins were incubated for 60 min at 37 °C with
a reaction buffer containing 200 nm streptavidin to bind to biotin (B) to block one end of the DNA. In addition, p represents a 5′
phosphate, and the asterisk represents the radiolabel. A, 20 nm *HC101/102 was incubated with 50 nm Artemis (lane 2); 25
nm DNA-PKcs and 50 nm Artemis (lane 3); 50 nm Artemis and 100 nm X4·LIV (lane 4); and 25 nm DNA-PKcs, 50 nm Artemis,
and 100 nm X4·LIV (lane 5). B, *20 nm HC 101/102 was incubated with 20 nm pHC115/116 and 50 nm Ku, 25 nm DNA-PKcs,
25 nm Artemis, and 100 nm X4·LIV as indicated.

9

Does X4:LIV stimulate NHEJ-dependent joining of 3’ overhangs, 5’ overhangs, and blunt
ends?
In addition to evaluating X4:LIV-mediated stimulation of Artemis resection on unpaired
substrates, here I assess the ability of ligase to stimulate joining of substrate pairs. To probe the
effect of DNA end conformation on stimulation of joining, I used substrate pairs with different
end structures (overhang or blunt), biotinylated at one end for prevention of nuclease activity using
streptavidin and radiolabeled for visualization. Additionally, to measure the rate of joining, I
performed a time course by terminating the reaction at multiple time points in order to determine
the amount of ligation product formed as a function of time. If ligase is capable of stimulating
NHEJ-mediated joining through recognition of an available 3’-OH, I expected that ligation product
formation would increase using substrates with 3’ overhangs, but not 5’ overhangs, blunt ends, or
modified ends lacking this 3’-OH.

Does a 5’ phosphate on the antiparallel strand affects the rate of NHEJ?
Recent evidence obtained using single-molecule FRET suggests that bridging of DNA ends
before ligation is dependent upon X4:LIV, which uses the 5’ phosphate group as a “recognition
element” to allow for other factors within the NHEJ core complex to stabilize the ends (Reid et
al., 2017). While our lab has performed studies using a 5’ phosphate on the radiolabeled strand of
many structurally distinct substrates, we have not examined a 5’ phosphate on the unlabeled
antiparallel strand and the effect on ligation.
By testing a 5’ phosphate on the antiparallel strand of DNA substrates with different end
configurations, here I evaluate how joining of the unlabeled strand affects ligation of the visible
10

labeled strand, which will offer a more detailed understanding of the process by which NHEJ
proteins are recruited to and ligate DNA ends.
I used our biochemical reconstitution system to test the effects of a 5’ phosphate on the
unlabeled strand of substrates containing 5’ overhangs, 3’ overhangs, and blunt ends, as compared
to substrates containing a 5’-hydroxyl group (OH) on the unlabeled strand. Additionally, to test
the effects of a 5’ phosphate on the rate of NHEJ-mediated joining, I performed a time course, as
described previously. If pairing of DNA ends is improved by the presence of a 5’ phosphate due
to bridging by X4:LIV, I expected to find that substrates with a phosphate on the antiparallel strand
are ligated more efficiently than those lacking this element.

Does a nucleosomal context affect the rate of NHEJ-dependent joining of DNA ends between
nucleosomes?
The majority of genomic DNA is wrapped around histone octamers, underscoring the
biological relevance of the repair of DSBs found within these regions of DNA. Here I use our
biochemical reconstitution system to test NHEJ-mediated end joining between nucleosomes. I
used purified recombinant Xenopus laevis octamers containing core histones H2A, H2B, H3, and
H4 (Histone Source, Colorado State University, Fort Collins, CO). Reconstitution of histone
octamers with defined-sequence DNA fragments containing a 147-bp nucleosomal positioning
sequence was performed using a previously established salt dialysis protocol (Dyer et al., 2004). I
evaluated how the length of DNA protruding from the nucleosome affects joining. By performing
these tests, my aim was to characterize how NHEJ-mediated joining of “naked” DNA ends is
affected by the presence of proximal nucleosomes.

11

CHAPTER 2.
EXPERIMENTAL PROCEDURES

2.1 OLIGONUCLEOTIDES AND DNA SUBSTRATES
Oligonucleotides used in this study were synthesized by Integrated DNA Technologies, Inc.
(San Diego, CA). Oligonucleotides were purified using 8 or 12% denaturing PAGE and DNA
concentration was determined spectrophotometrically. 5′ end radiolabeling of oligonucleotides
was performed using [γ-
32
P]ATP (3000 Ci/mol) (PerkinElmer Life Sciences) and T4
polynucleotide kinase (New England Biolabs). 3′ end radiolabeling was performed using [α-
32
P]
thymidine triphosphate (3000 Ci/mol) (PerkinElmer Life Sciences) and TdT (Promega).
Termination of the 3′ end-labeling reaction was achieved by adding a 13-fold excess of unlabeled
2′,3′-dideoxythymidine to [α-
32
P]TTP. Unincorporated radioisotope was removed using Sephadex
G-25 spin columns (Epoch Life Science). Duplex DNA substrates were created by adding a 20%
excess of unlabeled oligonucleotide to the radiolabeled complementary strand. To ensure
hybridization and to reduce secondary structure formation, all substrates were heated at 95 °C for
5 min and cooled at room temperature for 3 h, then at 4 °C overnight. Sequences of
oligonucleotides used in this study are as follows: HC101, 5′-C*G*T* T*AA GTA TCT GCA
TCT TAC TTG ATG GAG GAT CCT GTC ACG TGC TAG ACT ACT GGT CAA GCG CAT
CGA GAA CCC CCC TTT TTT-3′; HC102, 5′-GGT TCT CGA TGC GCT TGA CCA GTA GTC
TAG CAC GTG ACA GGA TCC TCC ATC AAG TAA GAT GCA GAT ACT TAA CG-biotin-
3′; HC115, 5′-GAT GCC TCC AAG GTC GAC GAT GCA GAC ACT GAT ATA TGT ACA
GAT TCG GTT GAT CAT AGC ACA ATG CCT GCT GAA CCC ACT ATC G-3′; HC120, 5′-
biotin-CGA TAG TGG GTT CAG CAG GCA TTG TGC TAT GAT CAA CCG AAT CTG TAC
12

ATA TAT CAG TGT CTG CAT CGT CGA CCT TGG AGG CAT C-3′; HC121, 5′-C*G*T*
T*AA GTA TCT GCA TCT TAC TTG ATG GAG GAT CCT GTC ACG TGC TAG ACT ACT
GGT CAA GCG CAT CGA GAA CC-3′; HC127, 5′-A*T*T* A*CT ACG GTA GTA GCT ACG
TAG CTA CTA CCG TAG TAA T-3′; HC128, 5′-biotin-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′; CG03, 5′-C*G*T* T*AA GTA TCT GCA TCT TAC TTG ATG GAG GAT CCT GTC ACG
TGC TAG ACT ACT GGT CAA GCG CAT CGA GAA CCC CCC TTT TT-dideoxy-C-3′; CG04,
5′-biotin-CGA TAG TGG GTT CAG CAG GCA TTG TGC TAT GAT CAA CCG AAT CTG
TAC ATA TAT CAG TGT CTG CAT CGT CGA CCT TGG AGG CAT-dideoxyC-3′; CG11, 5′-
C*G*T* T*AA GTA TCT GCA TCT TAC TTG ATG GAG GAT CCT GTC ACG TGC TAG
ACT ACT GGT CAA GCG CAT CGA GAA CCC CCC TTT TTT TTT T-3′; CG12, 5′-TTT TTT
TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT
CAT CGA GAA CCC CCC TTT TTT-3′; SL23, 5′-T*T*T* T*TT TTG CCA GCT GAC GCG
CGT CAG CTG GC-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′; 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′; JG277, 5′-TTT TTT CCC CGT TAA GTA
TCT GCA TCT TAC TTG ACG GAT GCA ATC GTC ACG TGC TAG ACT ACT GGT CAA
GCG GAT CGG GCT CG-3′. Asterisks indicate phosphorothioate linkages.
Oligonucleotides comprising 147 or 175 nucleotides (nt) of the 601 nucleosome positioning
sequence were synthesized by Integrated DNA Technologies, Inc. (San Diego, CA).
Oligonucleotides were purified using 8% denaturing PAGE and DNA concentration was
determined spectrophotometrically. To generate single-stranded DNA, oligonucleotides were
13

ligated in a reaction containing equimolar amounts of oligonucleotides 1 and 2, 20% excess
scaffold oligonucleotide, and T4 ligase (New England Biolabs, Ipswich, MA). Single-stranded
DNA was purified using 5% denaturing PAGE and DNA concentration was determined
spectrophotometrically. 5’ end radiolabeling of single-stranded DNA was performed using [γ-
32
P]ATP (3000 Ci/mmol) (PerkinElmer Life Sciences) and T4 polynucleotide kinase (New
England Biolabs, Ipswich, MA). Unincorporated radioisotope was removed using Sephadex G-
25 spin columns. Duplex DNA substrates were created by adding a 20% excess of unlabeled
single-stranded DNA (generated as described above) to the radiolabeled complementary strand.
To ensure hybridization and to reduce secondary structure formation, duplex DNA was heated at
95°C for 5 minutes and cooled at room temperature for 3 hours, then at 4°C overnight (Figure
2.1). Sequences of oligonucleotides used in this study are as follows: 147 oligo 1, 5’-CTG GAG
AAT CCC GGT GCC GAG GCC GCT CAA TTG GTC GTA GAC AGC TCT AGC ACC GCT
TAA ACG CAC GTA CGC GCT G-3’; 147 oligo 2, 5’-phosphate-TC CCC CGC GTT TTA
ACC GCC AAG GGG ATT ACT CCC TAG TCT CCA GGC ACG TGT CAG ATA TAT ACA
TCC TGT-3’; 147 oligo 3, 5’-ACA GGA TGT ATA TAT CTG ACA CGT GCC TGG AGA
CTA GGG AGT AAT CCC CTT GGC GGT TAA AAC GCG GGG GAC AGC G-3’; 147 oligo
4, 5’-phosphate-CG TAC GTG CGT TTA AGC GGT GCT AGA GCT GTC TAC GAC CAA
TTG AGC GGC CTC GGC ACC GGG ATT CTC CAG-3’; 147 scaffold 1, 5’-ACG CGG GGG
ACA GCG CGT AC-3’; 147 scaffold 2, 5’-CGC ACG TAC GCG CTG TCC CC-3’; 175 oligo 1,
5’- CCT ATA CGC GGC CGC CCT GGA GAA TCC CGG TGC CGA GGC CGC TCA ATT
GGT CGT AGA CAG CTC TAG CAC CGC TTA AAC GCA CGT ACG CGC-3’; 175 oligo 2,
5’-phosphate-TG TCC CCC GCG TTT TAA CCG CCA AGG GGA TTA CTC CCT AGT CTC
CAG GCA CGT GTC AGA TAT ATA CAT CCT GTG CAT GTA TTG AA-3’; 175 oligo 3, 5’-
14

TTC AAT ACA TGC ACA GGA TGT ATA TAT CTG ACA CGT GCC TGG AGA CTA GGG
AGT AAT CCC CTT GGC GGT TAA AAC GCG GGG GAC AGC GCG-3’; 175 oligo 4, 5’-
phosphate-TA CGT GCG TTT AAG CGG TGC TAG AGC TGT CTA CGA CCA ATT GAG
CGG CCT CGG CAC CGG GAT TCT CCA GGG CGG CCG CGT ATA GG-3’; 175 scaffold
1, 5’- GCG GGG GAC AGC GCG TAC GT-3’; 175 scaffold 2, 5’- AAC GCA CGT ACG CGC
TGT CC-3’.

Figure 2.1. Assembly of blunt 5’ radiolabeled 601 DNA. Synthesized oligonucleotides comprising 147 or 175 nt of the Widom
601 nucleosome positioning sequence were purified using 8% denaturing PAGE and DNA concentration was determined
spectrophotometrically. To generate single-stranded DNA, oligonucleotides were ligated in a reaction containing equimolar
amounts of oligonucleotides 1 and 2 (abbreviated oligo 1 and oligo 2), 20% excess scaffold oligonucleotide, and T4 ligase.
Single-stranded DNA was purified using 5% denaturing PAGE and DNA concentration was determined spectrophotometrically.
5’ end radiolabeling of single-stranded DNA was performed using [γ-32P]ATP (3000 Ci/mol) and T4 polynucleotide kinase.
Unincorporated radioisotope was removed using Sephadex G-25 spin columns. Duplex DNA substrates were created by adding a
20% excess of unlabeled single-stranded DNA (generated as described for radiolabeled ssDNA) to the radiolabeled
complementary strand. Duplex DNA was heated at 95°C for 5 minutes and cooled at room temperature for 3 hours, then at 4°C
overnight.
Plasmids containing dinucleosome DNA were provided by the Kurumizaka lab (Kato et al.,
2017). The 342-bp sequence, containing two Widom 601 sequences separated by 42 bp, was
isolated from a pGEM-T Easy vector by digestion with EcoRV and purified using 5% native
PAGE. The dinucleosome DNA sequence is as follows: 5’-ATCGA GAATC CCGGT GCCGA
GGCCG CTCAA TTGGT CGTAG ACAGC TCTAG CACCG CTTAA ACGCA CGTAC
15

GCGCT GTCCC CCGCG TTTTA ACCGC CAAGG GGATT ACTCC CTAGT CTCCA
GGCAC GTGTC AGATA TATAC ATCCA GGCCT TGTGT CGCGA AATTC ATACT
CGAGC GGACC CTATC GCGAG CCAGG CCTGA GAATC CCGGT GCCGA GGCCG
CTCAA TTGGT CGTAG ACAGC TCTAG CACCG CTTAA ACGCA CGTAC GCGCT
GTCCC CCGCG TTTTA ACCGC CAAGG GGATT ACTCC CTAGT CTCCA GGCAC
GTGTC AGATA TATAC ATCCG AT-3’.

2.2 PROTEIN EXPRESSION AND PURIFICATION
The X4:LIV complex was expressed and purified using a baculovirus expression system in
High Five cells (ThermoFisher Scientific). XRCC4 and C-terminal His-tagged DNA ligase IV
recombinant baculoviruses were a gift from Dr. Dale Ramsden (University of North Carolina
School of Medicine, Chapel Hill, NC). High Five cells were co-infected with XRCC4 and DNA
ligase IV baculoviruses at an equal multiplicity of infection. Harvested cells were resuspended in
Ni-NTA binding buffer (50 mm NaH2PO4 (pH 7.8), 500 mm KCl, 0.1% Triton X-100, 20
mm imidazole (pH 7.8), 2 mm β-mercaptoethanol) supplemented with protease inhibitors, then
sonicated and centrifuged. The supernatant was applied to Ni-NTA-agarose resin (Qiagen).
X4:LIV was eluted with elution buffer (binding buffer with 500 mm imidazole). High-resolution
purification was performed using two-step column chromatography with the ÄKTAexplorer
system (GE Healthcare Life Sciences). Ni-NTA affinity-purified eluate was dialyzed against Mono
Q buffer A (50 mm Tris-HCl (pH 7.5), 150 mm KCl, 10% glycerol, 0.05% Triton X-100, 1
mm DTT, 0.2 mm PMSF), loaded onto a Mono Q 5/50 GL anion exchange column (GE Healthcare
Life Sciences), and eluted with a linear gradient to 30% Mono Q buffer B (Mono Q buffer A with
1 m KCl). Peak fractions were pooled, dialyzed against Mono S buffer A (50 mm Tris-HCl (pH
16

7.5), 50 mm NaCl, 10% glycerol, 1 mm EDTA, 1 mm DTT, 0.2 mm PMSF), loaded onto a Mono
S 5/50 GL cation exchange column (GE Healthcare Life Sciences), and eluted with a linear
gradient to 100% Mono S buffer B (Mono S buffer A with 750 mm NaCl). Peak fractions were
stored at −80 °C. Purified X4:LIV was found to be active and to have no detectable nuclease
contamination. Purification of Ku, DNA-PKcs, and Artemis was performed as previously
described (Ma et al., 2002; Pannicke et al., 2004; Goodarzi & Lees-Miller, 2004; Chang et al.,
2015). Briefly, the recombinant Ku70/80 complex was purified from High Five cells by Ni-NTA
affinity, dsDNA (oligo) affinity, and anion exchange chromatography. Endogenous DNA-PKcs
was purified from HeLa cells using a series of column chromatography steps including anion
exchange, cation exchange, dsDNA (oligo) affinity, and size exclusion chromatography. Artemis
was purified from Sf9 cells by Ni-NTA affinity and anion exchange chromatography. Ku, DNA-
PKcs, and Artemis were found to have no detectable nuclease contamination.

2.3 NUCLEASE ASSAY
In vitro nuclease assays were performed in a 10-μl reaction volume in NHEJ buffer (25 mm
Tris-HCl (pH 8.0), 75 mm KCl, 10 mm MgCl2, 1 mm DTT, 0.5 mm ATP, 10% PEG 8000, with
200 nm streptavidin when using biotinylated substrates). Biotinylated DNA was incubated with
excess streptavidin prior to the addition of proteins for efficient blocking of biotinylated DNA
ends. Reactions containing 40 nm 32P-radiolabeled DNA substrate, 50 nm Artemis, 25 nm DNA-
PKcs, and 100 nm X4:LIV were incubated at 37 °C for 60 min. Ladders were generated by
incubating 80 nm 32P-radiolabeled ssDNA with 0.4 milliunits/ml of snake venom
phosphodiesterase 1 (Sigma) at 37 °C for 10 min. Reactions were terminated by heat inactivation
at 95 °C for 10 min. DNA was extracted using phenol:chloroform, resolved using either 8 or 12%
17

denaturing PAGE, and detected by autoradiography. Nucleolytic activity was quantitated using
Quantity One 1-D analysis software (Bio-Rad).

2.4 LIGATION ASSAY
In vitro ligation time courses were performed in a 12-μl reaction volume in NHEJ buffer.
Reactions containing 20 nm
32
P-radiolabeled DNA substrate, 20 nm unlabeled substrate, 50
nm Ku, and 100 nm X4:LIV were incubated at 37 °C and 2-μl aliquots were removed at the
indicated time points. Markers were generated using the same conditions. Reactions were
terminated by heat inactivation at 95 °C for 10 min. DNA was extracted and detected and ligation
was quantitated as described above.

2.5 NHEJ ASSAY
NHEJ components were incubated with DNA substrates at 37 °C for 1 h. Markers were
generated under the same conditions. Reactions were terminated by heating at 95 °C for 10 min,
and samples were subsequently deproteinized using phenol-chloroform extraction. Extracted DNA
was resolved using 8% denaturing PAGE and detected by autoradiography. Ligation efficiency
was quantitated using Quantity One 1-D analysis software (Bio-Rad).

2.6 JUNCTION SEQUENCE ANALYSIS
DNA was visualized by exposing dried radioactive gels to an X-ray film overnight. Ligated
DNA products were eluted from the gel, and junction sequences were amplified from these
products using PCR primers HC105 and HC114. Amplified junction sequences were TA cloned
into pGEM-T Easy vectors (Promega) and transformed into electrocompetent DH10B cells.
18

Transformed cells were plated on Luria broth-agar/ampicillin/X-gal, and white colonies were
selected for sequencing.

2.7 NUCLEOSOME RECONSTITUTION ASSAY
Recombinant Xenopus histone octamers (Histone Source, Colorado State University, Fort
Collins, CO) and either radiolabeled mononucleosome DNA or unlabeled dinucleosome DNA
were combined in a 10 ul volume containing 2 M NaCl. Several ratios of octamer to DNA were
tested to determine the optimal ratio for minimal remaining free DNA after reconstitution. A
serial salt dilution protocol as described in Dyer et al., 2004 was used to assemble nucleosomes.
Briefly, a stepwise reduction of the salt concentration was performed by the addition of 10 mM
Tris-HCl (pH 7.5) at room temperature at the following time points and volumes: 30 min, 10 ul;
1 h 30 min, 5 ul; 2 h 30 min, 5 ul; 3 h 30 min, 70 ul. Reactions were then incubated at room
temperature for 30 min and stored at 4°C. To assess reconstitution efficiency, nucleosomes and
free DNA were separated using 4% native PAGE and detected by autoradiography or SYBR
Green I staining. The amount of remaining free DNA was calculated by dividing free DNA
signal by the total signal in the lane. Reactions containing <5% free DNA were used in NHEJ
and ligation assays. Imaging analysis was completed using Quantity 1-D and ImageLab software
(Bio-Rad).

2.8 MONONUCLEOSOME NHEJ ASSAY
Biochemical purified NHEJ assays were performed in a 10 ul reaction volume in NHEJ buffer
(25 mM Tris-HCl (pH 8.0), 75 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.5 mM ATP, 10% PEG
8000). Reactions containing combinations of 25 nM radiolabeled DNA or mononucleosome
19

substrate, 25 nM Ku, 50 nM X4:LIV, 12.5 nM Artemis, and 12.5 nM DNA-PKcs were incubated
in a water bath at 37°C for 60 minutes. Reactions were divided in half for analysis by two methods.
To assess nucleosome stability under reaction conditions, reactions were immediately resolved
using 4% native PAGE and free DNA was detected and quantified as described above. To assess
NHEJ efficiency, reactions were boiled at 95°C for 10 mins and DNA was isolated using
phenol:chloroform extraction followed by ethanol precipitation. DNA was resolved using 5%
denaturing PAGE and detected using autoradiography. Ligation efficiency was calculated by
dividing the added signals of all ligation products by the total signal in the lane. Artemis activity
is indicated by a decrease in ligation product formation attributable to two aspects: Artemis activity
at the labeled 5' end results in an overall loss of signal in the lane, and Artemis activity at the 3'
end results in a decrease in substrate due to the generation of resection products. While material in
the wells of native gels is visible in lanes containing strongly DNA-binding proteins (Ku and
histones), this has no measurable effect on NHEJ assays.

2.9 DINUCLEOSOME LIGATION ASSAY
Biochemical purified dinucleosome ligation assays were performed in a 50 ul reaction
volume in NHEJ buffer (25 mM Tris-HCL (pH 8.0), 75 mM KCl, 10 mM MgCl2, 1 mM DTT,
0.5 mM ATP, 10% PEG 8000). Reactions containing combinations of 25 nM DNA or
dinucleosome substrate, 25 nM Ku, and 50 nM X4:LIV were incubated in a water bath at 37°C
for 60 minutes. To assess ligation efficiency, reactions were boiled at 95°C for 10 mins and
DNA was isolated using phenol:chloroform extraction followed by ethanol precipitation. DNA
was resolved using 5% denaturing PAGE and detected using SYBR Green I staining. Ligation
efficiency was calculated by dividing the signal of ligation product by the total signal in the lane.
20

Quantitative analysis was performed using Image Lab software (Bio-Rad).

21

CHAPTER 3.
EFFECTS OF DNA END CONFIGURATION ON XRCC4:DNA
LIGASE IV AND ITS STIMULATION OF ARTEMIS ACTIVITY

ABSTRACT
In humans, NHEJ is the major pathway by which DSBs are repaired. Recognition of each
broken DNA end by the DNA repair protein Ku is the first step in NHEJ, followed by the iterative
binding of nucleases, DNA polymerases, and the X4:LIV complex in an order influenced by the
configuration of the two DNA ends at the break site. The endonuclease Artemis improves joining
efficiency by functioning in a complex with DNA-PKcs that carries out endonucleolytic cleavage
of 5′ and 3′ overhangs. Previously, we observed that X4:LIV alone can stimulate Artemis activity
on 3′ overhangs, but this DNA-PKcs-independent endonuclease activity of Artemis awaited
confirmation. Here, using in vitro nuclease and ligation assays, we find that stimulation of Artemis
nuclease activity by X4:LIV and the efficiency of blunt-end ligation are determined by structural
configurations at the DNA end. Specifically, X4:LIV stimulated Artemis to cut near the end of 3′
overhangs without the involvement of other NHEJ proteins. Of note, this ligase complex is not
able to stimulate Artemis activity at hairpins or at 5′ overhangs. We also found that X4:LIV and
DNA-PKcs interfere with one another with respect to stimulating Artemis activity at 3′ overhangs,
favoring the view that these NHEJ proteins are sequentially rather than concurrently recruited to
DNA ends. These data suggest specific functional and positional relationships among these
components that explain genetic and molecular features of NHEJ and V(D)J recombination within
cells.
22

INTRODUCTION
DSBs are common events in multicellular eukaryotes, occurring at a rate of ∼10 DSBs/cell/day
(Lieber, 2010; Lieber & Karanjawala, 2004; Martin et al., 1985). These breaks can arise
physiologically, generated by RAG proteins in V(D)J recombination, or pathologically, from
causes including IR, oxygen free radicals, and inadvertent enzymatic action (Lieber, 2010;
Santivasi & Xia, 2014; Chance et al., 1979; Karanjawala et al., 2002; Ashour et al., 2015;
Mahowald et al., 2008). In diploid yeast, DSBs are resolved through HR, which takes place during
late S and G2 phases when a homology donor can be used for HDR. Nonhomologous DNA end-
joining (NHEJ) does not depend on the presence of a homology partner and occurs throughout the
cell cycle, making it the predominant pathway utilized for repair of DSBs in vertebrate somatic
cells (Renkawitz et al., 2014; Symington & Gautier, 2011).
NHEJ is initiated when Ku, a heterodimer of Ku70 and Ku80, is recruited to the site of a DSB,
allowing for subsequent recruitment of the Artemis endonuclease; DNA-PKcs; X4:LIV; DNA
polymerases μ and λ; and accessory factors XLF/Cernunnos and paralog of XRCC4 and XLF
(PAXX), as needed (Griffith et al., 1992; West et al., 1998). The flexibility of the NHEJ pathway
is reflected in the capacity of core factors to act iteratively and in any order on a variety of
structurally and chemically diverse DNA ends, contingent upon which steps are necessary for a
break to be resolved (Gu & Lieber, 2008).
Artemis is the only nuclease required for NHEJ, although other nucleases (e.g. PALF) may be
involved (Li et al., 2011). Alone, Artemis can act as a 5′ exonuclease (Li et al., 2014). Artemis
complexed with autophosphorylated DNA-PKcs has endonuclease activity and hairpin opening
activity (Ma et al., 2002; Niewolik et al., 2006; Gu et al., 2010). Recent evidence indicates that
23

NHEJ-mediated repair of DSBs is dependent upon the DNA end structural configuration.
Importantly, the Artemis·DNA-PKcs nuclease and X4:LIV are sufficient for processing and
ligation of overhangs with short regions (≤4 nt) of internal microhomology (Chang et al., 2016).
It was observed, however, that X4:LIV and Artemis, without DNA-PKcs, generates both cleavage
and ligation products using DNA with 3′ overhang ends; the addition of Ku does not affect this
activity. Further testing confirmed that this was not a result of transient annealing between two
DNA substrates, but a DNA-PKcs-independent nuclease activity of Artemis that occurs in the
absence of another DNA end (Chang et al., 2016).
Here, we investigate the mechanism by which Artemis acts as an endonuclease on dsDNA
without DNA-PKcs through the involvement of X4:LIV. We find that Artemis is recruited by
X4:LIV to specific DNA end structural configurations, namely a protruding 3′-OH at an overhang,
where the interaction of X4:LIV with Artemis permits nucleolytic action. We also discuss how
this DNA-PKcs-independent Artemis activity is compatible with the known genetics and
molecular biology of NHEJ.

RESULTS
X4:LIV stimulates Artemis endonuclease activity on 3′ overhangs without DNA-PKcs
Previously, we have observed that X4:LIV alone is capable of stimulating Artemis action on a
10-nt 3′ overhang (Chang et al., 2016). Artemis has intrinsic 5′ exonuclease activity on ssDNA,
but requires activation by DNA-PKcs to endonucleolytically cleave 3′ ends (Niewolik et al., 2006).
To confirm the DNA-PKcs-independent endonuclease activity, we incubated a radiolabeled 74-bp
duplex DNA substrate containing a 14-nt 3′ overhang with Artemis alone, Artemis and DNA-
PKcs, Artemis and X4:LIV, or all three (Figure 3.1). To minimize cutting of transiently denatured
24

DNA at the 5′ end of the top strand, phosphorothioate linkages were incorporated into the first 5
nt. Furthermore, 3′ biotinylation of the bottom strand was used to prevent action of Artemis at this
same DNA end. (We have found that using streptavidin to suppress protein binding or enzyme
action at biotinylated ends is not 100% effective and that the Artemis·DNA-PKcs complex can
overcome this, as demonstrated in the present study by the generation of a low level of 5′ cleavage
products.) As expected, we observe Artemis activity at the 3′ overhang in the presence of DNA-
PKcs (Figure 3.1, lanes 3 and 5). More importantly, we found that Artemis indeed cuts the 3′
overhang with a higher efficiency in the presence of X4:LIV than it does alone (Figure 3.1, lane 2
versus 4). These data indicate that Artemis is stimulated by X4:LIV for endonuclease activity on
a 3′ overhang in the absence of DNA-PKcs.

Artemis requires DNA-PKcs for cleavage of 5′ overhangs
We wondered if Artemis activity on 5′ overhangs would also be stimulated by X4:LIV
alone (i.e. without DNA-PKcs). To test this, we incubated a radiolabeled 72-bp duplex DNA
substrate containing a 10-nt 5′ overhang with proteins, as described (Figure 3.2). As expected,
Artemis alone is not able to cleave the 5′ overhang (Figure 3.2, lane 2). Artemis, in the presence
of DNA-PKcs, acts on the 5′ overhang with a high efficiency, comparable with that observed for
3′ overhangs (Figure 3.1, lanes 3 and 5 versus Figure 3.2, lanes 3 and 5). However, the
endonucleolytic cutting efficiency of Artemis in the presence of X4:LIV is not greater than that
of Artemis alone (Figure 3.2, lane 2 versus 4). These data show that Artemis activity on 3′
overhangs can be stimulated by X4:LIV, but is not stimulated for 5′ overhangs.

25

FIGURE 3.1. X4:LIV stimulates Artemis nuclease action on a 3′ overhang DNA end. 50 nm Artemis, 100 nm X4:LIV, and 25
nm DNA-PKcs were incubated with 40 nm *CG11/HC102 at 37 °C for 60 min. DNA was incubated with 200 nm streptavidin prior
to the addition of NHEJ proteins to suppress protein binding or enzyme action at the biotinylated DNA end. Ladders (lane L) were
generated by incubating 0.4 milliunits/ml of snake venom phosphodiesterase with 80 nm *CG12 at 37 °C for 10 min. DNA was
resolved using 8% denaturing PAGE. The first 5 nt of the top strand contain phosphorothioate linkages. The asterisk indicates a
32
P
radiolabel, B indicates biotin, and SA indicates streptavidin. Cleavage efficiency was calculated by dividing the signal of cleavage
products by the total signal in each lane. Efficiency values were normalized to background signal in the substrate-only lane. This
figure is representative of three gels from very similar experiments. The same basic result was observed in numerous additional
experiments with other preparations of X4:LIV. Cleavage efficiency mean ± S.E. were calculated for each of five conditions
(substrate only (DNA); Artemis (Art); Artemis and DNA-PKcs (Art+PK); Artemis and X4:LIV (Art+XL); and Artemis, DNA-
PKcs, and X4:LIV (Art+PK+XL)). A Student's t test was used to determine p values for differences in cleavage efficiency between
Art and Art + XL conditions, where p < 0.05 indicates significance. The graph represents the statistical analysis for the 3′ overhang
substrate CG11/HC102, where p = 0.02.
26

Figure 3.2. X4:LIV does not stimulate Artemis nuclease action on a 5′ overhang DNA end. 50 nm Artemis, 100 nm X4:LIV,
and 25 nm DNA-PKcs were incubated with 40 nm *JG277/HC128 at 37 °C for 60 min. DNA was incubated with 200
nm streptavidin prior to the addition of NHEJ proteins to suppress protein binding or enzyme action at the biotinylated DNA end.
DNA was resolved using 8% denaturing PAGE. The asterisk indicates a
32
P radiolabel, B indicates biotin, and SA indicates
streptavidin. Cleavage efficiency was calculated by dividing the signal of cleavage products by the total signal in each lane.
Efficiency values were normalized to background signal in the substrate-only lane. This figure is representative of three gels from
very similar experiments. Statistical analysis was performed as previously described. The graph represents the statistical analysis
for the 5′ overhang substrate JG277/HC128, where p = 0.74.

X4:LIV alone does not stimulate Artemis hairpin nicking activity
As Artemis and X4:LIV alone (i.e. without DNA-PKcs) cut at 3′ overhangs, but not at 5′
overhangs, we wondered if X4:LIV could stimulate Artemis hairpin nicking activity, which is
27

required for opening DNA hairpins formed at coding ends during V(D)J recombination. To test
this, we used a 20-bp blunt-ended hairpin substrate (Figure 3.3). Artemis preferentially nicks 2 nt
3′ of the hairpin tip, where steric constraints on these base pairs result in the formation of a single-
stranded/double-stranded DNA (ss/dsDNA) boundary. This activity generates 4-nt 3′ overhangs
that may be further processed for downstream ligation (Ma et al., 2002). We found that these
nicked hairpin products are formed in the presence of DNA-PKcs, but not X4:LIV alone (Figure
3.3, lanes 3 –5). Interestingly, although X4:LIV alone does not stimulate Artemis hairpin nicking
activity, we found that the addition of X4:LIV to Artemis and DNA-PKcs results in an increase in
hairpin nicking, as well as the generation of additional hairpin cleavage products (Figure 3.3, lane
3 versus 5). This may be due to the formation of a 4-nt 3′ overhang upon hairpin nicking by the
Artemis·DNA-PKcs complex, which allows for interaction of X4:LIV with the overhang via the
terminal 3′-OH and, as a result, increased stimulation of Artemis activity at this end. These data
show that X4:LIV does not stimulate Artemis activity on hairpins.
28

Figure 3.3. X4:LIV does not stimulate Artemis nuclease action on a DNA hairpin. 50 nm Artemis, 100 nm X4:LIV, and 25
nm DNA-PKcs were incubated with 40 nm *HC127 at 37 °C for 60 min. DNA was resolved using 12% denaturing PAGE. The
first 5 nt of the substrate contain phosphorothioate linkages. The asterisk indicates a
32
P radiolabel. The arrows indicate major and
minor cleavage sites for the generation of hairpin cleavage products, which are predominantly 21 to 23 nt from the radiolabel.
Cleavage efficiency was calculated by dividing the signal of cleavage products by the total signal in each lane. Efficiency values
were normalized to background signal in the substrate-only lane. This figure is representative of three gels from very similar
experiments. Statistical analysis was performed as previously described. The graph represents the statistical analysis for the hairpin
substrate HC127, where p = 0.14.

A 3′-OH at a 3′ overhang is required for X4:LIV stimulation of Artemis
Recognizing that X4:LIV alone stimulates Artemis at 3′ overhangs, but not at 5′ overhangs or
hairpins, we wondered if an available 3′-OH is essential for this stimulation to occur. To test this,
we used a 74-bp substrate similar to that used in our first test, but replaced the 3′ terminal
deoxynucleotide in the overhang with a 2′, 3′-dideoxynucleotide.
29

Figure 3.4. X4:LIV does not stimulate Artemis activity on a 3′ overhang DNA end lacking a terminal 3′-OH. 50 nm Artemis,
100 nm X4:LIV, and 25 nm DNA-PKcs were incubated with 40 nm *CG03/HC102 at 37 °C for 60 min. DNA was incubated with
200 nm streptavidin prior to the addition of NHEJ proteins to suppress protein binding or enzyme action at the biotinylated DNA
end. Ladders (lane L) were generated by incubating 0.4 milliunits/ml of snake venom phosphodiesterase with 80 nm *CG12 at 37
°C for 10 min. DNA was resolved using 8% denaturing PAGE. The first 5 nt of the top strand contain phosphorothioate linkages.
The asterisk indicates a
32
P radiolabel, B indicates biotin, SA indicates streptavidin, and ddC indicates a 2′,3′-dideoxycytidine.
Cleavage efficiency was calculated by dividing the signal of cleavage products by the total signal in each lane. Efficiency values
were normalized to background signal in the substrate-only lane. This figure is representative of three gels from very similar
experiments. Statistical analysis was performed as previously described. The graph represents the statistical analysis for the 3′
overhang substrate containing a 3′ terminal dideoxycytidine CG03/HC102, where p = 0.42.
We again observed that cutting of the 3′ overhang occurred when DNA-PKcs was present
(Figure 3.4, lanes 3 and 5). However, the cutting efficiency of Artemis and X4:LIV was not
substantially greater than that of Artemis alone (Figure 3.4, lane 2 versus 4). Fold-changes in
30

cleavage efficiencies in reactions containing DNA-PKcs are comparable with those observed for
the same reactions in our first experiment (Figure 3.1, lanes 3 and 5 versus Figure 3.4, lanes
3 and 5), indicating that Artemis cutting in the presence of X4:LIV alone can be attributed to an
interaction between X4:LIV and the 3′-OH, and not a change in the enzymatic activity of Artemis.
To determine whether the recessed 5′-OH on the unlabeled bottom strand contributes to this
DNA-PKcs-independent activity, we added a nonradioactive 5′-PO4 here and found that X4:LIV
stimulation of Artemis was unaffected by this feature of the DNA end (Figure 3.5).

Figure 3.5. Stimulation of Artemis by X4:LIV is not affected by a recessed 5’ phosphate group at a 3’ overhang DNA end.
50 nM Artemis, 100 nM X4:LIV, and 25 nM DNA-PKcs were incubated with 40 nM *HC101/pHC102 at 37°C for 60 minutes.
DNA was incubated with 200 nM streptavidin prior to the addition of NHEJ proteins in order to suppress protein binding or enzyme
action at the biotinylated DNA end. Ladders were generated by incubating 0.4 mU/ml snake venom phosphodiesterase with 80 nM
*CG12 at 37°C for 10 minutes. DNA was resolved using 8% denaturing PAGE. The first 5 nt of the top strand contain
phosphorothioate linkages, indicated by S. The asterisk indicates a [
32
P]-radiolabel, B indicates biotin, SA indicates streptavidin,
31

and P indicates a nonradioactive phosphate group. Cleavage efficiency was calculated by dividing the signal of cleavage products
by the total signal in each lane. Efficiency values were normalized to background signal in the substrate-only lane. This figure is
representative of three gels from very similar experiments.

A 3′-OH but not a 5′-PO4 at both DNA ends is critical for X4:LIV ligation activity
We wondered if certain chemical features of DNA ends were being utilized as recognition
elements for X4:LIV, allowing it to bind these ends and then recruit Artemis for endonucleolytic
action. If so, we posited that the addition or removal of X4:LIV recognition elements would affect
the rate of ligation independent of Artemis or DNA-PKcs. Recent evidence shows that DNA end
chemistry, particularly a 5′-PO4, indeed acts as a recognition element for X4:LIV, serving to
stabilize bridging of broken DNA ends (Reid et al., 2017).
32

Figure 3.6. A second 5′ phosphate group is not essential for ligation of two blunt DNA ends. 50 nm Ku and 100 nm X4:LIV
were incubated with 20 nm radiolabeled duplex DNA (*HC121/HC102 or *HC121/pHC102) and 20 nm pHC115/HC120 at 37 °C
for 90 min, and ligation efficiency was measured at the specified time points. DNA was incubated with 200 nm streptavidin prior
to the addition of NHEJ proteins to suppress protein binding or enzyme action at the biotinylated DNA ends. Markers (lane M)
were generated by incubating *JG163/JG166 for 60 min under the same conditions. DNA was resolved using 8% denaturing PAGE.
The first 5 nt of the radiolabeled top strand contain phosophorothioate linkages. The asterisk indicates a
32
P radiolabel, B indicates
biotin, SA indicates streptavidin, P indicates a 5′-phosphate group, and OH indicates a 3′- or 5′-hydroxyl group. Two bands denoted
by an asterisk are a result of self-dimerization and hairpinning of the radiolabeled duplex in a “head-to-head” or “tail-to-tail”
orientation. Ligation efficiency was calculated by dividing the signal of ligation products by the total signal in each lane. This
figure is representative of three gels from very similar experiments.
We have found that nuclease activity by Artemis is required for efficient ligation of non-
33

complementary DNA overhang ends, whereas only Ku and X4:LIV are required for efficient
ligation of blunt ends (Chang et al., 2016). To test the effect of the 5′-PO4 on ligation of DNA ends
independent of Artemis, we incubated a blunt-ended DNA duplex containing a 5′-PO4 on the top
strand with either of two radiolabeled blunt-ended duplexes, one of which contains a 5′-OH on the
bottom strand (Figure 3.6, lanes 1 –5) and the other a 5′-PO4 at this site (Figure 3.6, lanes 6 –10).
The “outside” DNA end of each duplex was biotinylated to prevent the formation of large multimer
ligation products. Time course assays containing Ku, which is required for efficient blunt-end
ligation (Chang et al., 2016), and X4:LIV revealed that the addition of a 5′-PO4 to the unlabeled
bottom strand does not stimulate ligation (Figure 3.6, lanes 6–10). In fact, the presence of both 5′-
PO4 groups results in a lower ligation efficiency than does a 5′-PO4 at only one of the two DNA
ends (Figure 3.6). The removal of the 3′-OH from the unlabeled bottom strand, however, results
in a decrease in ligation of the top strand to almost undetectable levels (Figure 3.7, lanes 6 –10).
This shows that both 3′-OH groups play a critical role in the ligation of two blunt DNA ends,
whereas a second 5′-PO4 is not necessary. Overall, these data demonstrate the effect of DNA end
configuration on Artemis stimulation and blunt-end ligation by X4:LIV, unifying both of these
related roles of X4:LIV in DNA end processing.
34

Figure 3.7. Two 3′-OH are required for ligation of two blunt DNA ends. 50 nm Ku and 100 nm X4:LIV were incubated with
20 nm *HC121/HC102 and 20 nm unlabeled duplex DNA (pHC115/HC120 or pHC115/CG04) at 37 °C for 90 min, and ligation
efficiency was measured at the specified time points. DNA was incubated with 200 nm streptavidin prior to the addition of NHEJ
proteins to suppress protein binding or enzyme action at the biotinylated DNA ends. Markers (lane M) were generated by incubating
*JG163/JG166 for 60 min under the same conditions. DNA was resolved using 8% denaturing PAGE. The first 5 nt of the
radiolabeled top strand contain phosphorothioate linkages. The asterisk indicates a
32
P radiolabel, B indicates biotin, SA indicates
streptavidin, P indicates a 5′-phosphate group, OH indicates a 3′- or a 5′-hydroxyl group, and H indicates a 3′ hydrogen. Ligation
efficiency was calculated by dividing the signal of ligation products by the total signal in each lane. This figure is representative of
three gels from very similar experiments.

The X4:LIV complex interferes with Artemis·DNA-PKcs action at 3′ overhangs
We observed across all of the Artemis nuclease assays that the sum of all cleavage products
(both 5′ and 3′) was greater for reactions containing the X4:LIV complex relative to those
35

containing only Artemis and DNA-PKcs. To examine this in more detail, we quantitated 3′ and 5′
cutting efficiencies separately in the assays where these cleavage products are both generated
(i.e. assays containing 3′ overhangs). We found that there are more 3′ than 5′ cleavage products
generated in reactions containing Artemis and DNA-PKcs without X4:LIV. However, the addition
of X4:LIV results in a decrease in 3′ products and an increase in 5′ products (Figure 3.7, lane 3
versus 5). This observation is consistent for both 3′ overhang substrates (Figure 3.1 and 3.4).
To confirm this, we radiolabeled the 3′ end of a 74-bp duplex containing a 10-nt 3′ overhang
and incubated with proteins as previously described (Figure 3.8). We found that Artemis and
DNA-PKcs generate more 3′ cleavage products than do Artemis, DNA-PKcs, and X4:LIV
combined. In this case, although X4:LIV would not recognize the terminal dideoxythymidine at
the 3′ overhang and would therefore not stimulate Artemis action at this DNA end, the
Artemis·DNA-PKcs contribution accounts for cutting in lanes containing Artemis, DNA-PKcs,
and X4:LIV (Figure 3.8, lane 5). These data indicate that there is some interference between
X4:LIV and DNA-PKcs with respect to Artemis action at 3′ overhangs.
36

Figure 3.8. X4:LIV and Artemis:DNA-PKcs do not simultaneously occupy a 3′ overhang DNA end. 50 nm Artemis, 100
nm X4:LIV, and 25 nm DNA-PKcs were incubated with 40 nm HC101/HC102* at 37 °C for 60 min. DNA was incubated with
200 nm streptavidin prior to the addition of NHEJ proteins to suppress protein binding or enzyme action at the biotinylated DNA
end. DNA was resolved using 12% denaturing PAGE. The asterisk indicates a
32
P radiolabel, B indicates biotin, SA indicates
streptavidin, and ddT indicates a 2′,3′-dideoxythymidine. Cleavage efficiency was calculated by dividing the signal of cleavage
products by the total signal in each lane. Efficiency values were normalized to background signal in the substrate-only lane. This
figure is representative of three gels from very similar experiments.
37

DISCUSSION
Previously, we have observed that Artemis cuts DNA overhangs in the presence of X4:LIV
and absence of DNA-PKcs (Chang et al., 2016). This finding challenged our current understanding
of the necessity of DNA-PKcs acting in complex with Artemis to support endonuclease activity.
Here, using in vitro nuclease assays, we find that X4:LIV alone is sufficient to stimulate Artemis
endonuclease activity specifically at 3′ overhangs containing a terminal OH. Analysis of the
X4:LIV complex reveals the importance of the 3′-OH, but not the 5′-PO4 of the same DNA end,
for joining of blunt ends. We surmise, then, that the 3′-OH is not only necessary for ligation but
also serves as a critical contact point for X4:LIV, where it can recruit Artemis, which can then act
locally in the absence of DNA-PKcs.

DNA end chemical configuration serves as a X4:LIV recognition element
Mammalian DNA ligases I, III, and IV share features conserved in all DNA and RNA ligases;
the nucleotidyl transferase and oligonucleotide/oligosaccharide binding-fold domains comprise
the core catalytic region. An additional N-terminal DNA-binding domain found in the three
mammalian DNA ligases is required for ligation activity (Pascal et al., 2004; Ellenberger &
Tomkinson, 2008). Analysis of the crystal structure of DNA ligase I suggests that a 3′-OH is
important for proper alignment of DNA ends in a nicked substrate. Both binding sites for divalent
metal ions are coordinated such that they may position the 5′-PO4 for efficient nucleophilic attack
by the 3′-OH (Pascal et al., 2004).
38

Figure 3.9. A model for stimulation of Artemis activity by X4:LIV. A, DNA-PKcs-independent Artemis activity, stimulated by
X4:LIV, occurs specifically at 3′ overhangs containing a terminal 3′-OH. B, Artemis is auto-inhibited by an interaction between its
N-terminal catalytic domain and residues Asn-456, Ser-457, and Glu-458 (indicated by red circles) within its C-terminal tail
(Niewolik et al., 2017). X4:LIV interacts with the DNA ligase IV interaction region of C-terminal Artemis at residues 485–495
(indicated in yellow) (De Ioannes et al., 2012; Malu et al., 2012). The interaction between X4:LIV and Artemis releases the
autoinhibitory tail from the catalytic domain, allowing Artemis nuclease activity to occur. C, upon the generation of a DNA double-
strand break, X4:LIV is recruited to a 3′ overhang DNA end through recognition of a terminal 3′-OH. Artemis interacts with X4:LIV
(described in B) and can act locally at 3′ overhangs. D, Artemis can process 3′ overhang DNA ends with either DNA-PKcs or
X4:LIV. Red arrows indicate major and minor cut sites. The Artemis·DNA-PKcs complex preferentially cuts 4 nt 3′ of the
ss/dsDNA boundary, with minor products generated near the major cut site. Artemis stimulated by X4:LIV acts locally,
preferentially cutting 1 nt into the overhang end, with minor products generated 5′ of the major cut site, extending further into the
overhang.
Our finding that X4:LIV stimulation of Artemis occurs only at 3′ overhangs supports the role
of the 3′-OH as a critical contact point for X4:LIV, as this effect depends upon the availability of
a sterically accessible 3′-OH not present in 3′ terminal dideoxynucleotides, 5′ overhangs or hairpins
(Figure 3.9A). This is further substantiated by the decrease in ligation activity observed upon the
removal of a 3′-OH, but not a 5′-PO4, at one DNA end. The disparity between these results and
39

recent single-molecule fluorescence resonance energy transfer data, which suggests that it is the
5′-PO4 that serves as the critical recognition element (Reid et al., 2017), can be accounted for by
differences in which chemical reaction steps are being measured. Although single-molecule
fluorescence resonance energy transfer was utilized for the evaluation of bridging of
complementary 4-nt 3′ overhangs, here we measure covalent ligation of blunt ends. It is possible
that bridging of the overhangs, which are subject to transient annealing, requires both 5′-
PO4 groups for stabilization. In contrast, the 3′-OH may be more critical for catalytic activity by
X4:LIV to occur. This suggests that the catalytic step is rate-limiting rather than the noncovalent
bridging step.
Interestingly, we find that having a 5′-PO4 at both DNA ends results in a reduction in ligation
efficiency as compared with a 5′-PO4 at only one end (Figure 3.7). This result might be attributed
to repulsion between the negatively charged 5′-PO4 groups, making it less likely for the ends to be
correctly aligned for ligation.

A model for X4:LIV stimulation of Artemis endonuclease activity
A recent study suggests a model of Artemis autoinhibition in which residues Asn-456, Ser-
457, and Glu-458 within the C-terminal tail associate with the N-terminal catalytic region,
preventing substrate binding and inhibiting nuclease activity (Niewolik et al., 2017). Artemis also
contains a putative X4:LIV interaction domain at residues 485–495 within its C-terminal tail (De
Ioannes et al., 2012; Malu et al., 2012). Consistent with our findings using full-length Artemis, a
truncated Artemis mutant lacking a portion of the C-terminal tail is not stimulated for endonuclease
activity at the 3′ overhang (data not shown). We propose that, after binding to a 3′ overhanging
DNA strand via a 3′-OH recognition element, X4:LIV recruits Artemis to the DNA end; the ligase
40

complex may then activate Artemis by occupying the C-terminal X4:LIV-binding domain, thereby
obstructing the interaction between the C-terminal tail residues and the N-terminal catalytic region
(Figure 3.9B). This model provides an explanation for targeting of Artemis to specific DNA end
configurations as well as DNA-PKcs-independent endonuclease activity in the presence of X4:LIV
specifically at 3′ overhang ends (Figure 3.9C).
In addition to our finding that Artemis may be stimulated in the absence of DNA-PKcs, we
also observe that X4:LIV stimulation of Artemis generates cleavage products closer to the 3′-OH.
The Artemis:DNA-PKcs endonuclease preferentially cleaves 3′ overhangs 4–6 nt 3′ of the
ss/dsDNA boundary (Ma et al., 2002). Here, we similarly find that the addition of DNA-PKcs
results in cutting 3–6 nt 3′ of the ss/dsDNA boundary, with the major product at 4 nt (Figure 3.9D).
Interestingly, Artemis and X4:LIV (without DNA-PKcs) cut 1 nt 5′ of the 3′-OH, with minor
products being generated as cutting extends further into the overhang (Figure 3.9D). These
observations are consistent with our model, where binding of X4:LIV at the 3′-OH directs Artemis
to cut near that DNA end.
Recent single-particle cryo-electron microscopy data indicates that bridging of two ends at a
DSB is facilitated by a synaptic complex comprising Ku, X4:LIV, and XLF (Chen et al., 2021). It
was proposed binding of the X4:LIV-XLF scaffold complex (consisting of two molecules of
X4:LIV bridged by the XLF homodimer) at DSB allows for the recognition and sealing of both
nicks sequentially by each X4:LIV complex.

Physiological relevance of DNA-PKcs-independent Artemis endonuclease activity in V(D)J
recombination
In wild-type pre-B or pre-T cells, during V(D)J recombination, each coding end typically
41

suffers nucleolytic removal of 1 to 10 bp, contributing to the junctional diversification (Gauss &
Lieber, 1996). The Artemis·DNA-PKcs complex is likely responsible for this action because this
nuclease is already present at coding ends for hairpin opening. In wild-type pre-B or pre-T cells,
the signal DNA ends only rarely suffer nucleotide loss (Gauss & Lieber, 1996).
In mutant mammalian cells lacking DNA-PKcs kinase activity (e.g. rodent mutants, such as
murine SCID, or other SCID animals, such as equine SCID (Meek et al., 2008)), the signal ends
often suffer more signal end nucleolytic processing than in wild-type cells (Kulesza & Lieber,
1998; M. R. Lieber, Hesse, Mizuuchi, et al., 1988). There is substantial genetic and biochemical
evidence indicating that Artemis is the nuclease responsible for this processing, even in the absence
of DNA-PKcs (Touvrey et al., 2008). Some of this signal end processing may be due to the 5′
exonucleolytic action of Artemis, and this would leave a 3′ overhang. However, much of the signal
end processing in these DNA-PKcs mutants extends further, and there has been no explanation for
such deep nucleolytic processing (M. R. Lieber, Hesse, Lewis, et al., 1988). Our data here indicate
that the endonucleolytic activity of Artemis could occur at these signal ends due to partial
stimulation by the X4:LIV complex specifically at the 3′ overhangs generated by initial 5′
exonuclease action by Artemis. This type of activity would explain how processing by Artemis
can extend so deeply into signal ends in mammalian pre-B and pre-T cells in the absence of DNA-
PKcs.
In addition to explaining end processing during V(D)J recombination when DNA-PKcs is
absent in mutant cells, our studies are also relevant to the coding end processing in wild-type pre-
B and pre-T cells. Hairpin cleavage typically generates a 4-nt 3′ overhang with a terminal 3′-OH.
These studies show that this configuration is suitable for further processing by Artemis upon
stimulation by either DNA-PKcs or X4:LIV.
42

Physiological relevance of DNA-PKcs-independent Artemis endonuclease activity in NHEJ
When mammalian cells are subjected to IR or chemical agents that cause DSBs, cells lacking
DNA ligase IV are the most vulnerable. Cells lacking DNA-PKcs are sensitive, but not as sensitive
as cells lacking DNA ligase IV (Rooney et al., 2002). The results in our study show that Artemis
is partially activated at a subset of DNA end configurations (3′ overhangs) through recognition of
the 3′-OH by the X4:LIV complex and, therefore, is able to contribute to the endonucleolytic
processing of a subset of DSBs even in the absence of DNA-PKcs.
Of relevance to the wild-type situation, when DNA-PKcs is not competing with X4:LIV at a
3′ overhang, Artemis has its highest potential for nuclease activity. The reduction in
Artemis·DNA-PKcs cutting when X4:LIV is present at the 3′ overhang suggests that the ligase
complex and this nuclease complex cannot occupy the same 3′ overhang at the same time. Such
sequential action agrees with our biochemical reconstitution joining data (Ma et al., 2004) and our
data from junctions formed within cells during V(D)J recombination (Gauss & Lieber, 1996).

43

CHAPTER 4.
POLYMERASE ACTIVITY OF CHIMERIC TERMINAL
DEOXYNUCLEOTIDYLTRANSFERASE

ABSTRACT
The Pol X polymerases Pol μ, Pol λ, and TdT all play important roles in NHEJ owing to their
differentials level of template dependence. A structural element referred to as Loop1 is important
for modulating the level of template dependence: in TdT, which has primarily nontemplated
activity, Loop1 is rigid, whereas Loop1 of Pol μ, which has both template-dependent and -
independent activity, is disordered. To stabilize and solve the structure of the disordered Pol μ
Loop1, a chimeric TdT containing Pol μ Loop1 (TdT-μ) was constructed. Here we demonstrate
the biological relevance of TdT-μ, as well as the functional relevance of Loop1, by showing that
the characteristic template-dependence of Pol μ is exhibited by TdT-μ. These data illustrate the
importance of Loop1 in the level of template-dependent activity by WT TdT or Pol μ at a DSB
junction. Specifically, the flexibility of Pol μ Loop1 allows for binding of the incoming DNA
template strand without steric hindrance.

RESULTS
Ligation of compatible or incompatible 3′ overhangs in the presence of full-length WT TdT,
TdT-μ chimera, and Pol μ
In Figure 4.1, we show that X4:LIV alone is sufficient for ligation of compatible 4-nt 3′
overhangs (lane 3, 53% efficiency), whereas the addition of Ku 70/80 ensures an even more
44

efficient (>88%) ligation (lanes 4 –7) (Fig. 4.1A). This reflects in vivo data showing that Ku 70/80
is not essential for ligation of overhangs containing at least 2 bp of microhomology, which can be
generated upon hairpin nicking during V(D)J recombination (McElhinny et al., 2005). Sequencing
data show that ligation proceeds without nucleotide addition by a polymerase, likely because rapid
base pairing of these overhangs occurs faster than template-independent or template-dependent
polymerase activity at the DNA ends (Fig. 4.1B).
45

Figure 4.1. Ligation experiments in the presence of X4:LIV and Ku 70/80. A, comparative functional properties of TdT WT,
Pol μ, and TdT-μ chimera using complementary or noncomplementary overhangs. B, sequences of the products of ligation for
compatible overhangs. Note that only the top strand of sequence is shown. The junctions are where the Additions column is located,
and when no nucleotides are present here, this means that the left and right ends are joined directly, using the compatible
overhangs. C, sequences of the products of ligation for incompatible overhangs. Note that only the top strand of sequence is shown.
46

Whereas X4:LIV is sufficient for ligation of compatible overhangs, we find that substantial
ligation of incompatible 3′ overhangs does not occur in the absence of TdT-WT, Pol μ, or TdT-μ
chimera (lanes 12 –14) (Fig. 4.1A). Sequencing data show that at least 1 bp of microhomology
must become available through either template-dependent or template-independent nucleotide
addition before ligation of the ends can occur (Fig. 4.1C). In particular, these data show that full-
length TdT adds nucleotides randomly until at least 1 nucleotide is available for base pairing with
the downstream strand. As expected, this reflects the known template-independent activity of TdT-
WT. Conversely, Pol μ adds nucleotides mainly template-dependently, although there are four
instances where a template-independent addition of 1 nucleotide occurs prior to templated
addition, illustrating a small degree of template-independent activity (Fig. 4.1C). Importantly, the
activity of TdT-μ chimera is much more like that of Pol μ than that of TdT in terms of template
dependence because most of the nucleotides added are A and thus complementary to the T
overhang of the right-hand DNA end. This clearly indicates that Pol μ Loop1 does indeed confer
template-dependent activity to the TdT-μ chimera across strands, in the context of a DNA synapsis.
In addition to the ligated product sequence, the ligation efficiency also emphasizes that the chimera
is more like Pol μ than TdT. Specifically, ligation in reactions with Pol μ and TdT-μ chimera are
very similar in efficiency, in contrast to the efficiency for the TdT reactions (Fig. 4.1A,
compare lanes 12, 13, and 14). Therefore, it is important to note that both the joining efficiency
and junctional sequencing support the conclusion that the chimeric protein behaves like Pol μ
rather than TdT.

DISCUSSION
Comparison of Pol X activities during NHEJ in the presence of different 3′ ends
47

The biochemical ligation tests using Ku 70/80, X4:LIV, and either the full-length TdT, Pol μ,
or TdT-μ chimera provide useful insights into the role of Pol X polymerases in NHEJ (Fig. 4.1).
First, TdT robustly adds nucleotides in a template-independent manner prior to ligation in the case
of incompatible DNA ends. However, TdT does not add nucleotides when compatible DNA ends
are being joined. This illustrates that the collision and annealing of the DNA ends is rapid relative
to the encounter of those ends with the Pol X polymerase. This observation confirms and extends
previous work showing that when DNA end structures are compatible, then new synthesis is
suppressed. This was indeed apparent in very early work before specific proteins were identified
for NHEJ (Thode et al., 1990; Roth & Wilson, 1986). More recently, the degree of Pol X
engagement was shown to be directly proportional to the extent to which there was a barrier to
direct ligation (due to sequence overhang incompatibility), both in vitro and in vivo, for Pol λ
(Waters et al., 2014).
Second, for incompatible DNA ends, Pol μ usually adds nucleotides that generate terminal
microhomology. But in ∼25% of instances in the experiments here for this configuration, it appears
that Pol μ adds at least 1 nucleotide in a template-independent manner. This raises the possibility
that the microhomology nucleotide is also template-independent, and we are only observing the
subset of events where Pol μ added, by chance, a nucleotide that provided 1 bp of terminal
microhomology. The remaining nucleotides could reflect fill-in synthesis by Pol μ in a template-
dependent manner. The clearest tests of template-dependent versus template-independent addition
by Pol μ are with dideoxynucleotides or immobilized DNA ends (Moon et al., 2015; Gu et al.,
2007; McElhinny et al., 2005), and in these tests, Pol μ shows both template-independent and
template-dependent activity. Our in trans structural studies show synthesis across a discontinuous
template by both Pol μ and TdT-μ chimera (Loc’h et al., 2016). All of the aforementioned
48

biochemical and structural data are consistent with the original conception of Pol μ's ability to
cross a discontinuous template (Gu et al., 2007).
Most importantly for this study, in the NHEJ biochemical assays using TdT-μ chimera, the
nucleotide additions are much more like those of Pol μ than of TdT. This illustrates the importance
of Loop1 in the distinction between TdT and Pol μ, directly in the context of NHEJ, and validates
that TdT-μ chimera can be used to characterize the role of Loop1 in Pol μ and the SD1 region at
the structural level.

49

CHAPTER 5.
NONHOMOLOGOUS DNA END JOINING OF NUCLEOSOMAL
SUBSTRATES IN A PURIFIED SYSTEM

ABSTRACT
The nonhomologous DNA end joining pathway is required for repair of most DSBs in the
mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining
of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate
ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a
manner dependent on the presence of free DNA flanking the NCP. Local resection at DNA ends
by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome
substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes,
dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state
imposes severe constraints on NHEJ nuclease and ligase activities.

INTRODUCTION
After a DSB is induced, repair by NHEJ is initiated when the Ku70/80 heterodimer (Ku) binds
to DNA ends at the break site. Ku binding is followed by the recruitment of core NHEJ factors as
necessary. These include Artemis, which acts as an endonuclease in complex with DNA-PKcs;
XRCC4:DNA ligase IV (X4:LIV), XLF, PAXX, and DNA polymerases µ and λ (Griffith et al.,
1992; West et al., 1998). The NHEJ pathway has the capacity to resolve a chemically and
structurally diverse set of breaks, owing to the iterative nature by which NHEJ factors process
50

DNA ends (Gu & Lieber, 2008; Chang et al., 2016). Though extensive investigation of NHEJ in
biochemical studies has provided a better understanding of joining of free ends in naked DNA,
little is known about how NHEJ proceeds in the context of chromatin.
Here, we investigate NHEJ in the context of chromatin by using blunt-ended mononucleosome
substrates in a purified biochemical system. Blunt ends were tested because they are the most
difficult DNA end configuration to join in the absence of a nuclease or polymerase, as there is
minimal possibility for transient base pairing of overhangs (Chang et al., 2016); therefore, these
ends would similarly pose a challenge to ligation in the chromatin environment of the cell, as well.
We find that ligation occurs in mononucleosomes containing 14 bp of blunt-ended DNA flanking
the NCP, suggesting that free DNA extending beyond the 147 bp directly contacting the octamer
surface is required for accessibility of the DNA ends. Dinucleosomal substrates lacking flanking
DNA behaved similarly. Further, we find that the Artemis:DNA-PKcs nuclease complex does not
act on the mononucleosome substrates tested, regardless of the presence of flanking DNA up to a
length of 14 bp. These studies provide essential information about NHEJ ligation and nuclease
action on defined nucleosomal substrates.

RESULTS
NHEJ-mediated ligation of mononucleosomes does not occur in the absence of free flanking
DNA
To investigate how NHEJ factors process different DNA end configurations, we have
established and currently utilize a purified biochemical NHEJ system in which ligation and local
(<20 bp) resection efficiencies of NHEJ reactions are quantitated using a gel-based assay. Briefly,
duplex DNA substrates with different end configurations (blunt, 5’ overhang, 3’ overhang, hairpin)
51

are incubated with NHEJ factors, and ligation and local resection efficiency of reactions are
measured by detecting substrate and product on a denaturing gel. This system has been used to
develop a model for how NHEJ factors work together to process and resolve different DNA
substrates (Chang et al., 2016). Here, we employ this biochemical system to compare the nature
and extent of NHEJ processing and joining of blunt-ended nucleosomal DNA versus naked DNA
of equivalent lengths.

Figure 5.1. Overview of nucleosome NHEJ assay. 5’ radiolabeled duplex DNA was assembled as previously described. DNA
and histone octamers were reconstituted into mononucleosomes using a serial salt dilution protocol (Dyer et al., 2004).
Mononucleosomes containing <5% free DNA were incubated with NHEJ proteins and reactions were halved and resolved using
two methods. Half of every reaction was immediately loaded on native polyacrylamide gels to determine nucleosome stability. The
remaining half of every reaction was deproteinized and loaded on denaturing polyacrylamide gels to determine ligation and
resection efficiency.
To generate mononucleosomal substrates, 5’ radiolabeled blunt-ended DNA molecules of
defined lengths comprised of the Widom 601 nucleosome positioning sequence (Lowary &
Widom, 1998) were reconstituted into nucleosomes using dilution assembly (Dyer et al., 2004)
(Figure 5.1). Increasing molar ratios of octamer:DNA were tested to determine the optimal ratio
52

for minimal residual free DNA after reconstitution (Figure 5.2). Nucleosomes containing <5% free
DNA were used in downstream assays (Figure 5.2, lane 4).

Figure 5.2. Determination of the optimal molar ratio of histone octamer to DNA for mononucleosome reconstitution.
Increasing amounts of histone octamer were incubated with a 5’ radiolabeled 147-bp fragment of the Widom 601 sequence (Lowary
& Widom, 1998). DNA and octamers were reconstituted into nucleosomes using serial salt dilution (Dyer et al., 2004). Reactions
were immediately loaded onto a 4% native polyacrylamide gel in 0.1X TBE and detected by autoradiography. The amount of
remaining free DNA after reconstitution was calculated by dividing the signal of free DNA by the total signal in each lane. Ratios
of octamer to DNA are as follows: Lane 1, 0.8:1; Lane 2, 1:1; Lane 3: 1.2:1; Lane 4: 1.4:1.
The high affinity of the histone octamer for duplex DNA suggests that steric constraints might
limit availability of nucleosome-associated DNA ends to binding and processing by NHEJ protein
complexes. To test this, we incubated NCPs containing no flanking DNA, or 147 bp of equivalent
naked DNA, with Ku, X4:LIV, Artemis, and/or DNA-PKcs. After incubation at 37°C for one hour,
reactions were halved for analysis by two methods. To assess nucleosome stability under reaction
conditions, half of every reaction was loaded directly on a native acrylamide gel. To assess NHEJ
53

efficiency, the remaining half of every reaction was deproteinized with phenol:chloroform and the
extracted DNA was loaded onto a denaturing gel. For 147-bp naked DNA, we find that the
presence of Ku, which has a high affinity for DNA ends, induces a shift in native conditions,
indicating protein binding and ligation product formation (Figure 5.3A). As observed in previous
studies (Chang et al., 2016), we also find that naked DNA reactions containing Ku and X4:LIV
exhibit the highest ligation efficiency, while the addition of DNA-PKcs results in a reduction of
this efficiency (Figure 5.3B, lanes 3 and 6). Mononucleosomes containing 147-bp DNA remain
stable under reaction conditions, as we do not observe any dissociation of DNA from octamers, as
would be indicated by an increase in free DNA (Figure 5.3C). Importantly, we confirm that ligation
is completely suppressed in mononucleosomes containing no flanking DNA (Figure 5.3D). These
data indicate that octamer-associated DNA ends in NCPs are not accessible to joining by Ku and
X4:LIV.
54

Figure 5.3. NHEJ-dependent ligation of mononucleosomes containing 147-bp DNA is completely suppressed. 25 nM Ku,
50 nM X4:LIV, 12.5 nM Artemis, and 12.5 nM DNA-PKcs were incubated with 25 nM radiolabeled 147-bp naked DNA or 25
nM radiolabeled mononucleosomes at 37 °C for 60 min. Reactions were resolved using two methods. Reactions containing naked
DNA were A, loaded directly on a 4% native polyacrylamide gel in 0.1X TBE, or B, deproteinized using phenol:chloroform
extraction and loaded on a 5% TBE-urea polyacrylamide gel in 1X TBE. Reactions containing mononucleosomes were C, loaded
directly on a 4% native polyacrylamide gel in 0.1X TBE, or D, deproteinized using phenol:chloroform extraction and loaded on a
5% TBE-urea polyacrylamide gel in 1X TBE. Bands above substrate (147 bp) in panel A indicate shifting of the DNA due to
protein binding, ligation product formation, or a combination of both. Ligation efficiency was calculated by dividing the signal of
ligation products by the total signal in each lane. To adjust for background noise, ligation efficiency of the substrate-only lane
was subtracted from the ligation efficiency of each lane. These gels are representative of several partial replicates and at least one
full replicate.

Naked DNA is ligated more efficiently than nucleosomal DNA of the same length
To investigate the requirement for accessible flanking DNA for ligation of mononucleosomes,
nucleosome substrates were assembled using 175 bp of the 601 sequence such that 14 bp of DNA
flank the histone octamer, 14 bp being the minimum length of duplex DNA necessary for stable
55

Ku binding (Yoo et al., 1999). (Positional characterization of the octamer in relation to the 601
sequence has been documented extensively in previous studies (Chua et al., 2012; Makde et al.,
2010; Vasudevan et al., 2010)). For 175-bp naked DNA, we observe shifts in the presence of Ku
(Figure 5.4A) and ligation efficiencies comparable to those of the same reactions containing 147-
bp naked DNA substrates (Figure 5.3B versus 5.5A). We also find that the 175-bp
mononucleosomal substrate, which is subject to translational phasing of the histone octamer along
the DNA (Sivolob & Khrapunov, 1995), is stable under reaction conditions (Figure 5.4B).

Figure 5.4. Mononucleosomes remain stable under reaction conditions. 25 nM Ku, 50 nM X4:LIV, 12.5 nM Artemis, and 12.5
nM DNA-PKcs were incubated with 25 nM radiolabeled 175-bp naked DNA or 25 nM radiolabeled mononucleosomes at 37°C for
60 min. Reactions containing either A, naked DNA or B, mononucleosomes were loaded directly on a 4% native polyacrylamide
gel in 0.1X TBE. In panel B, a low DNA signal (175 bp) indicates that mononucleosomal substrates do not dissociate under reaction
conditions. Bands migrating above the substrate band (175 bp in panel A and Nucleosome in panel B) indicate shifting of the
substrate due to protein binding, ligation product formation, or a combination of both. These gels are representative of several
partial replicates and one full replicate.
Further, we do observe ligation of these mononucleosomes (Figure 5.5B). Although the level
of ligation of mononucleosomes was lower than that of equivalent naked DNA reactions,
mononucleosomes are ligated in a manner similar to equivalent naked DNA (Figure 5.5A and
5.5B). Specifically, reactions containing Ku and X4:LIV are most efficiently ligated (Figure 5.5A
and 5.5B, lanes 3 and 7) and the addition of DNA-PKcs reduces these levels (Figure 5.5A and
56

5.5B, lane 6). This suggests that, though joining activity on nucleosomal DNA is moderately
suppressed, flanking DNA of only 14 bp at each duplex end is indeed sufficient for NHEJ-
dependent joining of mononucleosomes to proceed.

Figure 5.5. Mononucleosomes containing 175-bp DNA are less efficiently ligated than equivalent naked DNA. 25 nM Ku, 50
nM X4:LIV, 12.5 nM Artemis, and 12.5 nM DNA-PKcs were incubated with 25 nM radiolabeled 175-bp naked DNA or 25 nM
radiolabeled mononucleosomes at 37°C for 60 min. Reactions containing either A, naked DNA or B, mononucleosomes were
deproteinized using phenol:chloroform extraction and loaded on a 5% TBE-urea polyacrylamide gel in 1X TBE. Ligation efficiency
was calculated by dividing the signal of ligation products by the total signal in each lane. To adjust for background noise, ligation
efficiency of the substrate-only lane was subtracted from the ligation efficiency of each lane. *[Ligation efficiency calculated for
panel B, lane 8 would not reflect actual ligation efficiency. Artemis activity at the labeled 5' end results in an overall loss of signal
in the lane. Artemis activity at the 3' end results in formation of the doublet, affecting the value for total substrate in the analysis.]
These gels are representative of several partial replicates and at least one full replicate.

Nuclease activity by Artemis:DNA-PKcs is completely suppressed in nucleosomal DNA
To determine if the nucleosomal state suppresses nuclease activity by Artemis:DNA-PKcs, we
compared the formation of local DNA end products on naked DNA and mononucleosomes for
both 147-bp and 175-bp DNA. Nuclease activity is indicated by a decrease in observable ligation
product formation (due to loss of signal as a result of Artemis:DNA-PKcs resection at the 5’
radiolabeled end) and/or an increase in resection product formation (due to Artemis:DNA-PKcs
resection at the unlabeled end). For 147-bp and 175-bp naked DNA, Artemis:DNA-PKcs activity
is carried out as expected, resulting in the formation of resection products (Figure 5.3B and 5.4A,
57

lane 8). We do not observe Artemis:DNA-PKcs activity on mononucleosome substrates,
regardless of the presence of 14 bp of flanking DNA (Figure 5.3D and 5.4B, lane 8). These data
indicate that, unlike ligation, nuclease activity is completely suppressed in nucleosomal DNA at
these same lengths.

Dinucleosomes containing no free flanking are not ligated by Ku and X4:LIV
Several studies have shown that in addition to repositioning by ATP-dependent remodelers,
nucleosomes are susceptible to spontaneous thermal fluctuation of the octamer along the DNA
(Widom, 1999). One study suggests that nucleosome mobility through translational positioning
(translocation along the duplex) is necessary for accessibility of nucleosomal DNA to transcription
factors (Ura et al., 1995).
To determine if translational mobility affects accessibility of nucleosome-associated DNA
ends, we constructed a physiologically spaced dinucleosome substrate containing no free flanking
DNA (Figure 5.6). Here, two octamers are positioned on 342 bp of blunt-ended nucleosomal DNA
comprising two copies of the 601 sequence such that no free DNA extends beyond the outside
edge of either nucleosome core. A length of 48 bp of DNA between nucleosomes allows for
increased mobility of nucleosomes along the DNA, and, in turn, exposure of the previously
inaccessible nucleosome-associated DNA ends to Ku and X4:LIV. An increase in ligation signal
using these dinucleosome substrates would contrast with the absence of ligation of
mononucleosomes containing no free flanking DNA, supporting a role for translational mobility
in accessibility of nucleosomal DNA.
58

Figure 5.6. Determination of the optimal molar ratio of histone octamer to DNA for dinucleosome reconstitution. Increasing
amounts of histone octamer were incubated with a 342-bp fragment of DNA comprising two copies of the Widom 601 sequence
(17). DNA and octamers were reconstituted into nucleosomes using serial salt dilution (18). Reactions were immediately loaded
onto a 4% native polyacrylamide gel in 1X TBE and detected by SYBR Green I staining. The amount of remaining free DNA after
reconstitution was calculated by dividing the signal of free DNA by the total signal in each lane. Ratios of octamer to DNA are as
follows: Lane 1, 1.8:1; Lane 2, 2:1; Lane 3: 2.2:1.
As expected, 342-bp naked DNA is ligated highly efficiently (~38%) in the presence of Ku
and X4:LIV (Figure 5.7A, lane 2). Under the same reaction conditions, however, we do not
observe significant ligation of dinucleosomes (Figure 5.7B, lane 2). This supports our findings
above that NHEJ-mediated ligation of nucleosomes is suppressed in the absence of free flanking
DNA in a purified system using recombinant histones. Taken together these data suggest that Ku
and X4:LIV cannot access nucleosome-associated ends for joining and, furthermore, that
nucleosomal translocation does not occur to an extent sufficient for ligation by Ku and X4:LIV.
59

Figure 5.7. Dinucleosomes containing no free flanking DNA are not ligated by Ku and X4:LIV. 25 nM Ku and 50 nM X4:LIV
were incubated with A, 25 nM 342-bp naked DNA or B, 25 nM dinucleosome at 37 °C for 60 min. Reactions were deproteinized
using phenol:chloroform extraction, resolved on a 5% TBE-urea polyacrylamide gel in 1X TBE, and visualized using SYBR Green
I staining. Here we observe several substrate bands due to formation of three-dimensional conformations caused by foldback of the
dinucleosome DNA, which contains two direct repeats of the 601 nucleosome positioning sequence. [Resolution of 342-bp DNA
on strongly denaturing alkaline agarose gels shows a single substrate band (data not shown)]. Ligation efficiency was calculated
by dividing the signal of ligation products by the total signal in each lane. Gels are representative of three replicates.

DISCUSSION
Here, we use a well-established purified NHEJ system to examine how nucleosome substrates
are processed and joined by NHEJ factors. We find that the efficiency of nucleosome joining by
X4:LIV depends on the presence of free flanking DNA adjacent to the NCP. In mononucleosomes
containing no flanking DNA, no joining occurs (Figure 5.8). However, upon the addition of 14 bp
of DNA to either side of the core particle (175 bp total of nucleosomal DNA), ligation of
mononucleosomes proceeds, though less efficiently than, but in the same manner as it does on
equivalent naked DNA (Figure 5.9). In other words, combinations of NHEJ factors which ligate
naked DNA efficiently do the same for mononucleosomes, though at a 2.5 to 3-fold reduced level.
Furthermore, ligation of 175-bp nucleosomes but not 147-bp nucleosomes suggests that 14 bp of
DNA flanking the NCP is sufficient for loading of Ku and X4:LIV. The Artemis:DNA-PKcs
nuclease complex is unable to act on nucleosomal DNA ends, regardless of the presence of
flanking DNA of a length that is relevant to DSBs within the typical internucleosomal distance
60

(Figure 5.8 and 5.9). Overall, we find that, depending on the presence of flanking DNA and
therefore accessibility of DNA ends, the nucleosomal state suppresses to different extents ligation
and local resection by NHEJ proteins.

Figure 5.8. Summary of NHEJ of 147-bp substrates. Summarized are the ligation and resection efficiencies of NHEJ reactions
containing 25 nM Ku, 50 nM X4:LIV, 12.5 nM Artemis, and/or 12.5 nM DNA-PKcs, abbreviated respectively as Ku, X4:LIV, Art,
and PK. Efficiencies of reactions containing 25 nM 147-bp naked DNA and 25 nM 147-bp mononucleosomes are boxed in white
and gray, respectively. Minus signs (-) indicate less than 0.5% ligation or resection efficiency. Plus signs (+) indicate level of
ligation or resection efficiency in relation to the highest efficiency calculated for either substrate, where + indicates minimum
efficiency above 0.5%, ++ indicates intermediate efficiency, and +++ indicates maximum efficiency.
In contrast to SSBR and HR, NHEJ is a process in which events are limited to within ~20 bp
from each DNA end. In one interesting study, it was observed that Ku alone is able to load onto
nucleosome-associated DNA ends, and can translocate inward toward the dyad as far as 50 bp by
peeling the DNA away from the surface of the histone octamer (Roberts & Ramsden, 2007).
However, no enzymatic action (ligation or nuclease resection) was examined. Based on these
results, we anticipated that Ku binding would allow for ligation of 147-bp mononucleosomes.
However, our data show that in an NHEJ system capable of end joining, Ku does not enhance the
accessibility of nucleosome-associated ends to an extent that permits ligation.
61

Figure 5.9. Summary of NHEJ of 175-bp substrates. Summarized are the ligation and resection efficiencies of NHEJ reactions
containing 25 nM Ku, 50 nM X4:LIV, 12.5 nM Artemis, and/or 12.5 nM DNA-PKcs, abbreviated respectively as Ku, X4:LIV, Art,
and PK. Efficiencies of reactions containing 25 nM 175-bp naked DNA and 25 nM 175-bp mononucleosomes are boxed in white
and gray, respectively. Minus signs (-) indicate less than 0.5% ligation or resection efficiency. Plus signs (+) indicate level of
ligation or resection efficiency in relation to the highest efficiency calculated for either substrate, where + indicates minimum
efficiency above 0.5%, ++ indicates intermediate efficiency, and +++ indicates maximum efficiency.
As we observed using mononucleosome substrates, we find that NHEJ-mediated ligation of
dinucleosomes is suppressed in the absence of free flanking DNA. In addition to underscoring the
requirement for free DNA in accessibility of nucleosomes to joining by NHEJ factors, these
dinucleosome data suggest that octamer translocation-dependent exposure of nucleosome-
associated DNA ends does not occur. These findings are supported by a biochemical study of non-
ATP-dependent DNA accessibility in nucleosomes (Anderson et al., 2002). Investigation of
restriction enzyme activity on nucleosomes reconstituted using DNA of multiple lengths and
comprised of either natural or synthetic nucleosome positioning sequences showed that
translational mobility of nucleosomes is not a primary mechanism by which enzyme accessibility
is mediated. It was instead shown that enzymes access nucleosomes via transient dissociation of
62

DNA from the octamer, which, applied in the context of this study, is likely not sufficient for
efficient ligation of the nucleosome-associated ends.
Our findings indicate that activity by the Artemis:DNA-PKcs complex on nucleosomes is not
governed by the presence of flanking DNA. Instead, local resection at nucleosomal DNA ends is
completely suppressed. As indicated by reduced X4:LIV activity in mononucleosomes containing
flanking DNA, structural elements pose a barrier to enzyme activity in nucleosome complexes
independent of the positioning and presumed accessibility of DNA ends. A possible explanation
for complete suppression of local nucleolytic activity on mononucleosomes is a difference in
structural requirements for activity by the ligase and nuclease complexes. For NHEJ-mediated
endonuclease activity to proceed, a series of steps are likely dependent on the presence of free
DNA that is not wrapped around the surface of the octamer. Ku binding to the terminal 14 bp
usually precedes DNA-PKcs binding (Yoo et al., 1999). DNA-PKcs must then contact some
length of the DNA terminus to trigger autophosphorylation of itself in cis, and this
autophosphorylated DNA-PKcs is then able to activate the endonuclease activity of Artemis
(Goodarzi et al., 2006; Hartley et al., 1995). It may be that, compared to the smaller X4:LIV
complex (~180 kDa), 14 bp of DNA adjacent to the histone octamer may not allow for binding
and processing by the Artemis:DNA-PKcs complex (~547 kDa). Given the average
internucleosomal length of 40 bp in the vertebrate genome, the average distance from the edge of
an octamer to the midpoint between nucleosomes is 20 bp. Therefore, DSBs in the range we have
tested are relevant.
Our findings raise the question—how do NHEJ factors overcome steric challenges posed by
nucleosomes to process and resolve DSBs in the cell? There is broad genetic and cellular evidence
for the activation of DSB repair mechanisms which involve extensive nucleosome remodeling and
63

histone tail modification in chromatin surrounding the site of the break (Scully et al., 2019). In one
such study, the cryo-EM structure of SWI/SNF bound to a nucleosome is used to build a model of
chromatin remodeling by the SWI/SNF complex. Interestingly, these structural data indicate that
flanking DNA is required for formation of the stable SWI/SNF-nucleosome complex (Han et al.,
2020). These data combined with our results strongly suggest that ligation of DSB DNA ends at
an already accessible site is not sufficient for repair of most DSBs. It is likely that efficient repair
of DSBs in chromatin requires the recruitment of factors which expose an inaccessible break site
by means of nucleosome restructuring, and the elucidation of these mechanisms requires further
study, both in vitro and in vivo.

64

CONCLUDING REMARKS
Implications of these studies
At the onset of this project, members of our lab had designed a biochemical NHEJ assay in
order to investigate the NHEJ pathway in a purified system. This system was and continues to be
used to elucidate the activity and efficiency of NHEJ proteins on DNA ends of various structural
and chemical configurations, offering critical biochemical evidence to support existing genetic
insights, as well as bridging molecular and clinical data and helping to guide future endeavors to
target the NHEJ pathway in the development of cancer and disease therapies. This important work
was used as a foundation for addressing the questions conceived in this project. Up until now,
studies utilizing our biochemical NHEJ assay had only included naked DNA as substrates for
NHEJ proteins. In the cell, however, DNA is packaged into chromatin, a complex structure of
histone proteins and DNA which plays a pivotal role in the regulation of DNA organization,
transcription, and repair. The major aim of this project was to build on current knowledge of the
NHEJ repair pathway with our biochemical system, using both naked DNA and nucleosomal
substrates.
In a previous study we observed that Artemis alone was active in the absence of DNA-PKcs if
the X4:LIV complex was present. This had not yet been reported, as earlier studies showed that
the endonuclease activity of Artemis depended upon the presence of DNA-PKcs for activation of
the Artemis:DNA-PKcs complex. Here we find that Artemis resection of a 3’ overhang containing
a 3’-OH significantly increases upon the addition of the X4:LIV complex. This activity likely owes
to a previously proposed interaction between the self-inhibitory C-terminal region of Artemis and
the DNA binding domain of DNA ligase IV, causing the ligase complex to disrupt auto-inhibition
of Artemis activity by its own C-terminal tail and, in turn, activating Artemis. We find that this
65

activity does not occur on other DNA end structures (3’ dideoxynucleotides, 5’ overhangs,
hairpins). Additionally, we find that a 3’-OH group at each of two DNA termini is essential for
ligation of two blunt DNA ends, whereas only one 5’ phosphate (at either one of the two DNA
ends) is sufficient. Collectively these data suggest that DNA end chemistry serves as a recognition
element for the ligase complex, and, more broadly, supports the important role of end chemistry
in NHEJ processing indicated in previous studies.
Our studies into NHEJ-mediated repair of nucleosomal DNA ends largely suggest that
accessibility to NHEJ proteins is required for efficient joining or resection to proceed. In our
purified nucleosomal NHEJ assays, unmodified nucleosomes either with or without flanking blunt-
ended DNA (i.e., free DNA immediately adjacent to the NCP) can be studied for interaction with
Ku, X4:LIV, and/or Artemis under reaction conditions similar to those used for comparable naked
DNA. We find that ligation of nucleosomes only proceeds in the presence of at least 14 bp of
flanking DNA, while local Artemis resection does not occur on nucleosomal substrates with
overhangs of this length.

Preliminary foundations for future studies
The complex nature of chromatin is a result of the almost innumerable combinations of histone
modifications and variants required for versatile, efficient regulation of the genome. Using
dinucleosome and 12mer oligonucleosome substrates, I performed several pilot ligation
experiments testing the effect of ATP-dependent chromatin-remodeling complexes and histone
modifiers on accessibility of nucleosome-associated DNA ends (data not shown). These included
chromatin remodelers ATP-utilizing chromatin assembly and remodeling factor (ACF), Brahma
(BRM), and Brahma-related gene 1 (BRG1); and histone modifiers polycomb repressive complex
66

2 (PRC2), G9a, mixed lineage leukemia protein-1 (MLL1), and p300. We additionally conducted
experiments investigating phosphorylation of histone variant H2A.X by DNA-PKcs in
mononucleosomes containing 0 or 15 bp of flanking DNA. However, we have found no evidence
that these factors facilitate ligation or local resection of nucleosomal substrates.
A considerable amount of work is still needed to completely elucidate the effect of histone
modifications and variants—and the role of various histone modifiers and chromatin remodelers—
on accessibility to NHEJ proteins. My hope is that, as earlier studies did for this project, the studies
described in this thesis provide a foundation for future work on the NHEJ repair pathway,
particularly in the context of chromatin.
67

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Abstract (if available)

Abstract DNA double-strand breaks (DSBs) are a common occurrence in eukaryotic cells and are potentially the most seriously damaging to the genome because, if unrepaired, they can result in the loss of entire arms of chromosomes, containing thousands of genes. Nonhomologous DNA end-joining (NHEJ) and homologous recombination are major pathways for repair of these breaks. In vertebrates, all DSBs are pathologic except those generated during V(D)J recombination and class switch recombination. Pathologic breaks arise from multiple sources, including ionizing radiation, reactive oxygen species, and aberrant action by nucleases involved in normal cellular processes. NHEJ, the predominant pathway for repair of both physiologic and pathologic DSBs, involves several core components which are used to ligate a wide variety of DNA ends that differ in chemistry, sequence, and structure. The NHEJ core components have evolved to converge upon a theme of mechanistic flexibility which allows for the processing of such a variety of substrates. As in most DNA repair pathways, these components include a nuclease, polymerases, and a ligase complex, all of which, in addition to provisional accessory factors, are utilized for repair. Though the proteins of the NHEJ pathway have been genetically identified, a precise mechanistic understanding of this process has yet to be attained. For a better functional understanding, the components of NHEJ must be biochemically characterized. As detailed in this dissertation, I employed biochemical methods to study the NHEJ pathway using free DNA as well as nucleosomal substrates to gain an improved understanding of how NHEJ occurs at various DNA end configurations. More specifically, I tested the ability of the NHEJ ligase complex to stimulate the rate of NHEJ-dependent joining of different substrates. Additionally, I assessed the role of a 5’ phosphate in improving NHEJ efficiency. Finally, I used a biochemical NHEJ reconstitution system to determine how NHEJ is carried out in the context of chromatin. The insights gained from these studies contribute to our current functional model of NHEJ and may prove crucial in the development of DNA repair-targeted cancer therapies.

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Asset Metadata

Creator Gerodimos, Christina Anastasia (author)

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

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Cancer Biology and Genomics

Degree Conferral Date 2021-08

Publication Date 07/20/2022

Defense Date 06/09/2021

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

Tag chromatin,DNA damage,DNA repair,double-strand DNA breaks,NHEJ,nonhomologous DNA end-joining,nucleosome,OAI-PMH Harvest

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor An, Woojin (committee chair), Lieber, Michael (committee member), Zandi, Ebrahim (committee member)

Creator Email cgerodimos@gmail.com,gerodimo@usc.edu

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