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

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

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STUDIES ON THE ROLE OF ARTEMIS IN NON-HOMOLOGOUS DNA END-
JOINING TO UNDERSTAND THE MECHANISM AND DISCOVER THERAPIES

By

Howard Hoyon Chang

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
(Genetic, Molecular, and Cellular Biology)

December 2016

ii
AKNOWLEDGMENTS
I would like to thank my advisor, Dr. Michael Lieber for all the support he
has provided me during my time in his lab. It is amazing to think back on how
lucky I was to have a mentor that is involved in the actual experiments in the
various projects in the lab even though he is well established and a leader in the
DNA repair field. Because of this, I have always felt that I could talk to him about
experimental details that most mentors do not have time for.
In addition, I would like to thank my committee members for their guidance
and support throughout my graduate studies: Dr. Chih-Lin Hsieh and Dr. Ebrahim
Zandi. Dr. Hsieh was always available to help me with molecular biological
techniques that I was unfamiliar with and to also philosophize about life. Dr.
Zandi was helpful in providing input in protein purification optimization for our
high-throughput screen for Artemis inhibitors.
I also want to thank my former and present lab members that have
contributed to much needed moments of levity and fruitful discussions about lab
experiments: Dr. Xiaoping Cui, Dr. Rachel Britt, Dr. Noriko Shimazaki-Ishii, Dr.
Zhengfei Lu, Dr. Sicong Li, Dr. Zheng Zhang, Dr. Nicholas Punnunzio, Dr. Go
Watanabe, Zitadel Anne Esguerra, Shuangchao Ma, Christina Gerodimos, and
Cindy Okitsu-Yen.
I thank my parents, Hyon and Shinjae Chang for supporting my decision to
pursue this dream of mine, and to my siblings, Douglas and Hannah for visiting
and lending me much needed breaks from lab work.
iii
I would finally like to thank my wife, Michelle, for allowing me to pursue
and finish this part of my life. Halfway through my studies, we’ve had to live apart
for her to pursue her dreams of attending dental school. The time apart has been
more difficult than we could have imagined, and I will be forever grateful for her
love and support during that time.

iv
TABLE OF CONTENTS

Acknowledgements ii

List of Figures vi

Abstract ix

Chapter 1 GENERAL INTRODUCTION 1
1. Genomic instability and the role of Nonhomologous DNA End-joining 1
2. Identification and characterization of the factors in NHEJ 5
DSB Recognition 6
Resection by Artemis 8
DNA Synthesis During NHEJ and the Role of DNA Polymerases 11
DNA End ligation 13
3. Key Questions at the Inception of this Thesis Project 15

Chapter 2 EXPERIMENTAL DESIGN AND PREPARATION 18
1. Oligonucleotides 18
2. Protein expression and purification 21
3. Artemis-his purification for HTS drug screen 22
4. Biochemical nuclease assay 23
5. Reconstitution assay 24
6. Junction Sequence analysis 24

Chapter 3 HIGH THROUGHPUT SCREEN FOR INHIBITORS OF THE
ARTEMIS ENDONCULEASE FOR CANCER THERAPY:
OPTIMIZATION AND SCALING OF ARTEMIS
PURIFICATION FROM INSECT CELLS 26
Abstract 26
Introduction 27
Results 31
Discussion 44

Chapter 4 UNIFYING THE DNA END PROCESSING ROLES OF THE
ARTEMIS NUCLEASE: KU-DEPENDENT ARTEMIS
RESECTION AT BLUNT DNA ENDS 45
Abstract 45
Introduction 46
Results 47
Discussion 64

Chapter 5 STRUCTURE-SPECIFIC NUCLEASE ACTIVITIES OF
ARTEMIS AND THE ARTEMIS:DNA-PKCS COMPLEX 72
Abstract 72
Introduction 72
v
Unifying Elements of the Artemis Substrates 74
Contribution of DNA-PKcs to Substrate Recognition 78
Role of Artemis:DNA-PKcs in V(D)J Recombination 79
Roles of Artemis:DNA-PKcs in NHEJ 81
Future Directions 85

Chapter 6 DIFFERENT DNA END CONFIGURATIONS DICTATE
WHICH NHEJ COMPONENTS ARE MOST IMPORTANT FOR
JOINING EFFICIENCY 86
Abstract 86
Introduction 87
Results 89
Discussion 106

Concluding Remarks 113

List of Abbreviations 115

Bibliography 117

vi
LIST OF FIGURES

Figure 1.1 DSB can be generated by ROS and by replication errors 2

Figure 1.2 Pathological and physiological causes of DSBs in
mammalian cells
5

Figure 1.3 DNA-PK complex consists of Ku70/80 and DNA-PKcs 8

Figure 1.4 Artemis is the NHEJ nuclease 9

Figure 1.5 Three Pol X family members are involved in NHEJ 13

Figure 1.6 DNA Ligase IV complex 14

Figure 3.1 HTS assay is designed to find inhibitors of V(D)J
recombination 29

Figure 3.2 Codon optimized Artemis baculovirus can be expressed in
insect cells 32

Figure 3.3 Artemis-his purification scheme 33

Figure 3.4 Increasing the amount of Ni-NTA increases Artemis-his
binding 35

Figure 3.5 Artemis-his purified in denaturing conditions is not active 36

Figure 3.6 Ni2+ eluates include active Artemis-his 37

Figure 3.7 Triton X-100 (non-ionic detergent) does not inhibit Artemis-
his activity 39

Figure 3.8 Triton X-100 decreases multimeric Artemis-his complexes 41

Figure 3.9 Artemis-his purification results are reproducible from two
independent institutions 42

Figure 3.10 Precipitated Artemis-his was found under top filter of Mono
Q column 43

Table 3.1 Artemis-his purification summary 44

Figure 4.1 Optimal substrates for Artemis have ss/dsDNA boundaries
that direct Artemis to the point of preferred nuclease action 50

vii
Figure 4.2 Artemis activity comparison on blunt dsDNA and 5’ DNA
overhangs 51

Figure 4.3 Artemis activity on blunt dsDNA with dT ends 53

Figure 4.4 DNA-PKcs titration for Artemis activity on blunt ends 55

Figure 4.5 The Time course of 5’ endonuclease activity on blunt-ended
DNA reveals that Artemis resects up to 6-nt, requires DNA-
PKcs, and is modulated by Ku to preferentially cut 2-nt into
the duplex 58

Figure 4.6 DNA end breathing provides a substrate for Artemis 5’
exonuclease activity 59

Figure 4.7 The Time course of 3’ endonuclease activity on blunt-ended
DNA reveals that Artemis resects predominantly 1- to 3-nt,
requires DNA-PKcs, and is stimulated by Ku 61

Figure 4.8 Artemis 5’ end resection occurs before 3’ end resection and
progresses internally along the DNA with time 63

Figure 4.9 DNA end breathing model for Artemis action at blunt DNA
ends 67

Figure 5.1 Unifying Model Describing Artemis Activity on Common
Physiological DNA Substrates 75

Figure 5.2 Artemis Activity on Other DNA Substrates 77

Figure 5.3 Artemis Opens the Hairpins Generated in V(D)J
Recombination 80

Figure 5.4 Artemis is Involved in DNA End Repair via the NHEJ
Pathway 84

Figure 6.1 NHEJ reconstitution workflow 89

Figure 6.2 NHEJ of resection-dependent compatible 3’ overhang
requires Artemis and is strongly stimulated by Ku and DNA-
PKcs 91

Figure 6.3 Artemis resection of 3’ overhangs is stimulated by the
X4/LIV 92

viii
Figure 6.4 NHEJ of resection-dependent compatible 3’ overhangs is
strongly dependent on microhomology 94

Figure 6.5 The Ku, DNA-PKcs, Artemis, and X4/LIV complex is not
sufficient for NHEJ of incompatible DNA ends while blunt-
ended DNA only requires Ku and X4/LIV 95

Figure 6.6 NHEJ of 3’ incompatible ends is stimulated by Pol µ but not
Pol λ 98

Figure 6.7 PAXX only stimulates Blunt-ended NHEJ 99

Figure 6.8 NHEJ of 5’ overhang with a blunt-ended DNA is stimulated
by XLF and PAXX with no polymerase effect 101

Table 6.1 Sequence results of the most efficient NHEJ events 105

Table 6.2 NHEJ summary 106

Figure 6.9 Diagram of the end complex 107

ix
Abstract
Pathological DNA double-strand breaks (DSBs) are one of the most harmful
forms of DNA damage, since both of the DNA strands are broken. These breaks
can result in cell death from the loss of a chromosomal arm or by promoting the
apoptotic pathway. In mammalian cells, DSBs are repaired predominantly by the
nonhomologous DNA end-joining (NHEJ) pathway. Physiological DSBs are
created during V(D)J recombination and class-switch recombination (CSR) and
require the NHEJ pathway to resolve these breaks. Thus, defects in NHEJ result
in marked sensitivity to ionizing radiation and the ablation of lymphocytes. NHEJ
is typically imprecise, a characteristic that is useful for immune diversification in
lymphocytes, but which might also contribute to some of the genetic alterations
that cause aging and cancer. This negative feature is offset by the flexibility of
NHEJ proteins in handling many types of DNA end configurations and many
types of damage. Mechanistic studies have revealed that the NHEJ pathway can
be targeted for the treatment of cancers that utilize this pathway. Artemis is a
structure-specific NHEJ nuclease that is required to open the DNA hairpin
intermediates in V(D)J recombination. Thus, inhibiting Artemis can be used as a
first-in-class nuclease inhibitor for the treatment of blood cancers that rely of
V(D)J recombination, such as acute lymphoblastic leukemia (ALL). The
development of a high-throughput screen (HTS) for Artemis inhibitors has
required the scaling and optimization of Artemis purification. In addition, we use
biochemical assays to unify Artemis activity to propose a function-based model
x
along with the development of a direct gel NHEJ reconstitution assay to
determine the contribution of NHEJ factors in DSB repair.

1
CHAPTER 1
GENERAL INTRODUCTION

1.1 GENOMIC INSTABILITY AND THE ROLE OF NONHOMOLOGOUS DNA
END-JOINING
The mammalian genome not only encodes the blueprint for life, it also
presents a vast target for genotoxic agents. It is estimated that the mammalian
genome undergoes approximately 100,000 alterations per day (Rich et al., 2000).
These alterations can lead to DNA DSBs, and in fact there are an estimated ten
DSBs per day per cell based on early passage primary human and mouse
fibroblasts (Lieber, 2010; Lieber and Karanjawala, 2004; Martin et al., 1985).
DSBs represent one of the most dangerous forms of DNA damage, since having
unrepaired DSBs can lead to cell death from the loss of a chromosomal arm or
through the activation of the p53-mediated apoptosis cascade (Rich et al., 2000).
Pathological DSBs arise from ionizing radiation (IR), reactive oxygen
species (ROS), replication errors, and inadvertent cleavage by nuclear enzymes
(Lieber et al., 2003). The majority of IR that affects humans originates from the
decay of radioactive isotopes from within the earth and from cosmic radiation that
penetrate the atmosphere from space. These high-energy IR sources are able to
penetrate cells and generate free radicals by dislodging electrons from the water
molecules within cells (Figure 1.1A) (Ward, 1988). The ROS are also generated
as normal byproducts of oxidative metabolism. The mitochondria of respiring
2
cells convert approximately 0.1 – 1% of oxygen to superoxide anion radical (O
2
-
)
and subsequently hydroxyl radicals (OH) (Figure 1.1A) (Chance et al., 1979;
Karanjawala et al., 2002a). It is believed that approximately 10
9
ROS are
generated per cell per hour that can act as nucleophiles on the DNA bases and
phosphodiester backbone when in close proximity, and approximately one DSB
arises from 25 sites of single-strand DNA damage (Lieber, 2010). In addition to
damage by ROS, DNA enzymes are prone to errors. During DNA replication, the
DNA helicase in the replication fork may encounter a nick on one strand of the
DNA, which can cause the fork to collapse and result in a DSB (Figure 1.1B). In
addition, type II DNA topoisomerases create transient DSB in an ATP-dependent
manner to relieve tension in the DNA helix that is created during DNA replication
and gene expression (Lodish et al., 2000). Failures in topoisomerase to rejoin the
DNA ends can also result in permanent DSBs that must be repaired.

FIGURE 1.1. DSB can be generated by ROS and by replication errors. A, Water molecules
can become ionized from IR, which can then react with other water molecules to generate the
hydroxyl radical. The superoxide anion, which is a byproduct of respiration, can be transformed
into hydrogen peroxide. Hydrogen peroxide can then be reduced into the hydroxyl radical. If these
hydroxyl radicals are in created close to the DNA backbone, they can act as a nucleophile on the
DNA backbone to create DSBs. B, The migrating replication fork can collapse when it encounters
a nick, thereby creating a DSB.

3
DSBs can also be created by physiological processes in lymphocytes
during V(D)J recombination and CSR. V(D)J recombination is a process by which
immunoglobulin (Ig) and T cell receptor (TCR) variable domain exons are
constructed from V, D, and J sub-exonic coding elements in the pre-B and pre-T
lymphocytes, respectively. These sub-exonic elements are each flanked by a
recombination signal sequence (RSS) that consist of a heptamer (CACAGTG), a
12 or 23-bp spacer, and a nonamer (ACAAAACA) (Gellert, 1997; Schatz and
Swanson, 2011). Recombination activating gene proteins (RAG-1 and -2) form a
complex with High-mobility group box protein-1 (HMGB1) to form the RAG
complex, which binds at the RSS. The RAG complex nicks the 5’ end of the
heptamer at the border of each coding element and utilizes the 3’-OH on the
antiparallel strand as the nucleophile for transesterification in order to generate a
DNA hairpin on the coding element terminus and a blunt terminus at the RSS
(called a signal end) (van Gent et al., 1996). The formation of the DNA hairpin
creates a DSB that will be joined to another coding end (such as J) to form a
complete variable domain exon. The DNA hairpin formed at the coding end of
one gene element must be opened and joined to the DNA hairpin formed at the
other gene element in order to construct a functional V(D)J gene. While this
recombined V(D)J segment lies permanently upstream of a constant region in T-
lymphocytes in order for it to function as a membrane bound receptor, the V(D)J
segment of B-lymphocytes are initially upstream of the µ chain (Cµ), which allows
these cells to generate the IgM antibody isotype. Ig heavy chain CSR is then
required in mature B-lymphocytes for the Ig heavy chain Sµ region to break and
4
join with the switch region upstream of other constant regions (Cγ, Cα, or Cε) to
allow the cells to express the IgG, IgA, and IgE antibody isotypes, respectively
(Durandy, 2003). It has been shown that the process of switching the constant
region requires activation-induced cytidine deaminase (AID) in order to initiate a
lesion that leads to a nick at the switch regions, which leads to the formation of
DSB intermediates (Durandy, 2003). AID only initiates its C to U deamination
lesions in single-stranded DNA, which is provided by R-loops that form at the
switch regions (Yu et al., 2003). Once again, these DSB intermediates must be
re-joined to a new constant region for successful CSR.
These pathological and physiological processes require a DNA repair
pathway to repair these DSBs. In eukaryotic cells, two mechanistically unique
repair pathways have evolved: homologous recombination (HR) and NHEJ. HR
is a precise DNA repair pathway that requires a sister chromatid (or homologous
chromosome), and thus is limited to only the S and G2 phases of the cell cycle.
In contrast, NHEJ does not require a sister chromatid and can occur during the
whole cell cycle (Figure 1.2). DNA breaks may result in DNA ends with any of a
variety of configurations. The blunt, hairpin, 5’ or 3’ overhang ends, and DNA
lesions are processed by the NHEJ pathway in an error-prone manner to resolve
the break, with the joined junction being preferred to the potential loss of a
chromosomal arm.
5

FIGURE 1.2. Pathological and physiological causes of DSBs in mammalian cells. After
DSBs are created, the DNA can be repaired by two major pathways; NHEJ and homologous
recombination (HR). HR can occur during S and G2 phases of the cell cycle because there is a
sister chromatid present and in close proximity. The advantage of NHEJ is that it can occur during
the entire cell cycle, and thus is the most relevant for somatic cells.

1.2 IDENTIFICATION AND CHARACTERIZATION OF THE FACTORS IN
NHEJ
In NHEJ, the DSB is first recognized by Ku heterodimer (Ku70/80), which
acts as a ‘tool belt’ onto which other NHEJ proteins are recruited as needed.
When DNA resection is required, a DNA-dependent protein kinase catalytic
subunit (DNA-PKcs) is recruited in complex with Artemis. DNA-PKcs then
undergoes auto-phosphorylation and activates Artemis endonuclease activity.
Activated Artemis then gains the ability to cut various DNA end configurations at
the single- to double-strand (ss/ds) DNA boundaries. DNA polymerase X family
Physiological DSB

1. V(D)J recombina2on
2. Class switch recombina2on breaks
Pathological DSB

1. Ionizing radia2on
2. Reac2ve oxygen species
3. Replica2on across nicks
4. Inadvertent enzyme ac2on
5. Mechanical stress
HR
Late S and G2
NHEJ
En2re Cell cycle
Cleavage
phase
Repaired DNA
Repair
phase
6
members participate to add nucleotides. It is thought that the iterative resection
and addition steps continue until there is a region of microhomology (MH) that
forms to stabilize the DNA ends for ligation to occur by the DNA ligase complex
consisting of DNA ligase IV and XRCC4 (X-Ray repair cross-complementing
protein 4).

DSB Recognition
The Ku heterodimer protein complex was initially discovered as an auto-
antigen named after a scleroderma patient with the initials K.U. (Lieber, 2010).
The approximate size of the Ku complex based on gel mobility experiments was
70 and 80 kDa, and thus the complex was named Ku70/80 (Figure 1.3). It is
estimated that there are approximately 400,000 Ku molecules per cell (Anderson
and Carter, 1996). Genetic studies have shown that deficiencies in Ku70 or Ku80
do not cause embryonic lethality in mice but do consist of defects in growth,
V(D)J recombination, and premature cellular senescence (Ferguson and Alt,
2001). The 2.5 Å X-ray crystal structure of Ku70/80 in complex with DNA has
shown that Ku forms an asymmetric ring that binds to DNA-ends in a sequence-
independent manner (Walker et al., 2001). The structure also shows that the
channel of the ring is lined with positive charged residues that can accommodate
approximately two turns of dsDNA (~20 bp). These structural data correlated
strongly with the functional data suggesting that Ku interacts strongly with DNA
(K
D
= 5.9 x 10
-10
M) with a footprint of approximately 18 bp (de Vries et al., 1989;
West et al., 1998). The strong binding at the DNA ends not only protects the DNA
7
end from non-specific nuclease degradation, but it also acts to recruit proteins as
needed (Downs and Jackson, 2004). After binding, Ku translocates internally to
provide space for the other factors it recruits. Ku interacts with DNA-PKcs to form
the DNA-PK complex, which increases its association with DNA 10-fold (K
D
= 3.5
x 10
-11
M) (West et al., 1998). In addition, Ku70/80 interacts with Ligase IV to
promote ligation of DNA ends (Hsu et al., 2002).
DNA-PKcs is a 4,128 amino acid kinase with a molecular weight of 469
kDa (Figure 1.3). While not as abundant as Ku, there are approximately 50,000
to 100,000 DNA-PKcs molecules per HeLa cell (Anderson and Carter, 1996). It
binds to dsDNA ends of various configurations with an equilibrium dissociation
constant (K
D
) of 3 x 10
-9
M (West et al., 1998). Its association with dsDNA
increases 100-fold when Ku is present (West et al., 1998). DNA-PKcs is a
serine/threonine kinase in the phosphatidylinositol 3-kinase-related kinase (PIKK)
family that includes ATM (Ataxia telangiectasia mutated), which is also involved
in DNA repair. DNA-PKcs is the catalytic subunit of the DNA-PK complex, which
is comprised of Ku70/80 and DNA-PKcs. Binding of DNA-PKcs to dsDNA
induces autophosphorylation at ABCDE and PQR clusters. Phosphorylation at
the ABCDE cluster is thought to promote whereas phosphorylation of the PQR
cluster is though to inhibit DNA end processing. However, the effects of
autophosphorylation are complex and not well understood due to the shear
number of DNA-PKcs autophosphorylation sites (over 30 sites). DNA-PKcs also
phosphorylates Artemis, and Artemis in complex with DNA-PKcs is required to
open DNA hairpins formed as an intermediate in V(D)J recombination. Thus,
8
DNA-PKcs -/- cells are highly radiosensitive and have V(D)J recombination
deficiencies with DNA-PKcs mutant rodents displaying severe combined
immunodeficiency (SCID) (Meek et al., 2008). The first human DNA-PKcs mutant
patient was identified in 2009 with a classical T
-
B
-
lymphocyte SCID phenotype
(van der Burg et al., 2009). Current structural studies of DNA-PKcs have so far
yielded a low resolution 6.6 Å X-ray crystal structure (Sibanda et al., 2010).
Researchers are presently attempting to obtain structural information at a higher
resolution to better elucidate its interaction with DNA and other NHEJ factors.

FIGURE 1.3. DNA-PK complex consists of Ku70/80 and DNA-PKcs. The Ku heterodimer
consists of Ku70 and K80. They both consist of N-terminal von Willibrand A (vWA) domains, a
central core domain, nuclear localization signals (NLS), and a SAP (SAF-A/B, Acinus, and PIAS)
domain. DNA-PKcs is a 469 kDa PI3K kinase with a PQR and ABCDE phosphorylation cluster,
FAT and FAT-C (FRAP, ATM-TRRAP- C-terminal) domains.

Resection by Artemis
The action of a nuclease is often required to process broken ends for
polymerase action and subsequent ligation. While it is possible that DNA strand
cleavage will result in a 5’-phosphate and 3’-OH on the deoxyribose, there is
significant formation of uncommon moieties. For example, a 5’-aldehyde, 3’-
phosphoglycolate, 3’-phosphoglycoaldehyde, 3’-formyl phosphate, and 3’-keto-
9
2’deoxynucleotides that can prevent the binding and action of polymerases and
ligase (Povirk, 2012). In these instances, a nuclease is required to rejoin the DNA
ends.
In NHEJ, Artemis is the central nuclease involved in processing the ends
in conjunction with DNA-PKcs. Artemis is in the metallo-β-lactamase family of
nucleases characterized by conserved metallo-β-lactamase and β-CASP
domains (Figure 1.4). The β-CASP domain is aptly named after β-lactamase and
the four representative nucleases that contain this domain (CPSF, Artemis,
SNM1, and PSO2). This family contains prokaryotic and eukaryotic nucleases
that are believed to utilize a zinc-dependent mechanism to hydrolyze DNA or
RNA in various configurations (Dominski, 2007). Based on sequence
comparisons, it is believed that a conserved histidine motif (HxHxDH) is essential
in forming a coordination complex with the zinc ions for enzymatic activity
(Pannicke et al., 2004). Furthermore, mutations in two metallo-β-lactamases
(tRNase Z and Artemis) have been implicated in prostate cancer and SCID,
respectively (Dominski, 2007; Moshous et al., 2001).

FIGURE 1.4. Artemis is the NHEJ nuclease. Artemis contains an N-terminal β-lactamase
catalytic domain and a β-CASP domain shared by other members of the metallo-β-lactamase
family. The largely unstructured C-terminal tail is phosphorylated by DNA-PKcs.

Artemis
β-CASP C-term 692 aa β-lactamase domain
Proposed DNA-Lig IV interac;on
485 – 495
DNA-PKcs interac;on
402 – 403
DNA-PKcs phosphoryla;on sites
385 – 692
10
Artemis is also known as SNM1C, and is a homolog to SNM1A (Snm1)
and SNM1B (Apollo). SNM1 was initially discovered in S. cerevisiae as mutants
sensitive to nitrogen mustard, and the gene encoded an enzyme with the ability
to repair high molecular weight chromosomal DNA (Dominski, 2007). Both
Snm1A and Snm1B are involved in repairing inter-strand crosslinks. In contrast,
Artemis is involved in processing ssDNA at ss- and dsDNA boundaries. It is a
692-amino acid protein with a N-terminal β-lactamase domain and a C-terminal
tail. Artemis’ C-terminal region is phosphorylated by DNA-PKcs by way of its
interaction on aa 402-403 of Artemis (Lieber, 2010). In addition, recent structural
studies found that aa 485-495 interact in the hydrophobic pocket within α-helix 2
of DNA Ligase IV (De Ioannes et al., 2012).
Artemis alone has intrinsic 5’ exonuclease activity on ssDNA (Li et al.,
2014). On duplex DNA, Artemis, in complex with DNA-PKcs, has endonuclease
activity on the 5’ and 3’ DNA overhangs that are often created by pathological
DNA breaks and on DNA hairpins that are formed during V(D)J recombination.
Thus, patients lacking Artemis suffer from SCID due to a defect in antibody
formation (Moshous et al., 2001). In addition, a subset of T
-
B
-
NK
+
SCID which is
the result of an Artemis nonsense mutation at aa 192 has been found to occur
more frequently in Navajo and Apache Native Americans (Li et al., 2002).
We have determined biochemically that Artemis, in complex with DNA-
PKcs, resects 5’ and 3’ DNA overhangs in order to create DNA end structures
that are able to be ligated by the DNA ligase IV:XRCC4 complex (Lu et al., 2008;
Ma et al., 2002). At 5’ overhangs, Artemis cuts directly at the ss/dsDNA
11
boundary. However, when processing 3’ overhangs and DNA hairpins, it
preferentially leaves a 4-nt overhang (Ma et al., 2005a). Perfect DNA hairpins
have sterically-constrained tight turns, and thus have ss/ds boundaries at the last
2 base pairs (4-nts) (Blommers et al., 1989). This may make them structurally
similar to DNA overhangs. From these observations, we have proposed that
Artemis activity on duplex DNA can be unified under a model in which
Artemis:DNA-PKcs binds to the ss/dsDNA boundary to occupy 4-nt along the
single-stranded segment. This binding is followed by preferentially nicking on the
3’ side of those 4-nt. While the current model is extensive, it lacks information on
whether Artemis can cut blunt-ended DNA that can be generated by
chemotherapeutic agents, free radicals, or ionizing radiation (Povirk, 2012).

DNA Synthesis During NHEJ and the Role of DNA Polymerases
There are four mammalian polymerases in the Pol X family (Polymerase λ,
µ, β, and terminal deoxynucleotidyl transferase (TdT)); however only Pol λ, µ,
and TdT participate in NHEJ (Figure 1.5). Polymerase β is the only one lacking a
BRCA1 C-terminal (BRCT) domain, and thus participates in base excision repair.
POL4 is the only Pol X family member in S. cerevisiae, and is important in the
repair of 3’ overhangs (Wilson and Lieber, 1999). The finding that Pol µ forms a
complex with Ku and the DNA ligase complex was key in demonstrating that Pol
X family members participate in NHEJ (Mahajan et al., 2002). The main
functional difference of the polymerases is their ability to add nucleotides
independent of having a complementary template strand to interact with. TdT is a
12
template-independent polymerase that is only expressed in lymphocytes. Thus,
its role is limited to promote antigen receptor diversity during V(D)J
recombination. Conversely, Pol µ is expressed in all cells and has both template-
independent and template-dependent activity (Gu et al., 2007a). The phenotype
of Pol µ-deficient mice is impaired IgG light chain rearrangement, and thus
abnormal B-lymphocyte differentiation (Bertocci et al., 2003). Pol λ is primarily a
template-dependent polymerase, which also contains a functional lyase domain
in order to remove bases damaged by glycosylases (Lieber, 2010). Structural
studies on the Pol X family members have suggested that a flexible domain
named “loop 1” is utilized to substitute for a template strand in various degrees
depending on the polymerase (Juarez et al., 2006). In particular H329 within loop
1 of Pol µ was found to be important in conferring template-independent activity
(Moon et al., 2007).
13

FIGURE 1.5. Three Pol X family members are involved in NHEJ. A, Polymerase λ, µ, and TdT
are the polymerase X family members involved in NHEJ. B, Pol λ and µ are expressed in all cells
whereas TdT is expressed only in pre-B and T-lymphocytes.

DNA End ligation
The DNA ligase IV complex is responsible for the final ligation step of
NHEJ, and thus is essential (Grawunder et al., 1997; Wilson et al., 1997). The
complex consists of a 2:1 molar ratio of the 38 kDa XRCC4 protein to the 103
kDa DNA ligase IV protein according to gel filtration studies (Ahnesorg et al.,
2006) (Figure 1.6). DNA ligase IV is unstable in the absence of XRCC4, and
XRCC4-deficient cell lines are also deficient in ligase IV activity (Ellenberger and
Tomkinson, 2008). In addition, DNA ligase IV- and XRCC4-deficient cells show
508 aa
TdT
BRCT Nucleo,dyltransferase
575 aa
Pol λ
BRCT Nucleo,dyltransferase
494 aa
Pol μ
BRCT Nucleo,dyltransferase
Lyase
Lyase
Lyase
Ku:DNA interac,on
Ku:DNA interac,on
Pol X Expression Template-
dependent
Template-
independent
Pol λ All cells +++ +
Pol μ All cells ++ ++
TdT Pre-B/T lymphocytes - +++
A
B
14
severe neuronal growth defects and embryonic lethality (Ferguson and Alt,
2001). Interestingly, the Ku80/Ligase IV double null mouse is viable (Karanjawala
et al., 2002b). DNA ligase IV is distinguished from DNA ligase I and III by its
unique ability to join DNA ends that are non-complementary, making it the
preferred ligase for the error-prone NHEJ pathway (Gu et al., 2007a).
Functionally, DNA ligase IV activity is stimulated by the addition of Ku. The
stimulation is over 10-fold for DNA ends that only share 2-bp of MH and have a
1-nt gap to ligate over (Gu et al., 2007a). XRCC4 interacts with DNA ligase IV
through a linker region between the two BRCT domains on the C-terminus of
DNA ligase IV (Grawunder et al., 1998a). More recently, it has been claimed that
DNA ligase IV interacts with a short 11 aa fragment of Artemis (Malu et al., 2012;
Ochi et al., 2013). In all, these data strongly support the importance of the DNA
ligase IV complex in NHEJ.

FIGURE 1.6. DNA Ligase IV complex. The DNA ligase IV complex consists of DNA ligase IV,
along with XRCC4 and the more recently discovered factors, XLF (Cernunos) and PAXX.

15
XLF (XRCC4-like factor), also known as Cernunnos, and PAXX (Paralog
of XRCC4 and XLF) are the most recent factors to be involved in NHEJ (Figure
1.6). XLF was initially found in a T
-
B
-
SCID patient whose cells show IR
sensitivity and decreased V(D)J recombination (Dai et al., 2003). The 299 aa, 33
kDa XLF protein forms a heteromultimer with XRCC4 that promotes
XRCC4:Ligase IV-mediated ligation of incompatible DNA ends (Ahnesorg et al.,
2006; Gu et al., 2007b).
The 22 kDa PAXX protein was discovered using a bioinformatics
approach to find other members of structurally related proteins to XRCC4 and
XLF (Ochi et al., 2015). The C-terminus, and more specifically V199 and F201,
were found to be critical in its interaction with Ku70/80. It was also shown to
promote DSB repair at both cellular and biochemical levels (Ochi et al., 2015).

1.3 KEY QUESTIONS AT THE INCEPTION OF THIS THESIS PROJECT
There have been numerous efforts made to discover inhibitors of the
NHEJ pathway for the treatment of cancers. Traditionally, kinases have been a
popular target for small molecule inhibitors since phosphorylation is a reversible
major post-translational signaling mechanism that controls cellular processes.
However, the structurally similar active sites of kinases, makes these inhibitors
less specific. Attempts to develop DNA-PKcs inhibitors have been unsuccessful
due to high IC50 values that make it unsuitable in clinical settings. The NHEJ
polymerases of the Pol X family members (λ, µ, and TdT) can be targeted, but
they are not essential for NHEJ. More recently, a potential DNA ligase IV inhibitor
16
was discovered (Srivastava et al., 2012), but the purity and specificity of the
compound is in question, which may invalidate their results. Thus, Artemis,
remains the most specific target of NHEJ, since it is required to open the DNA
hairpin intermediates created during V(D)J recombination. Thus, the discovery
and subsequent development of an Artemis inhibitor would be a first-in-class
nuclease inhibitor for the treatment of acute lymphoblastic leukemia (ALL). To
that end we have screened an initial group of 30,000 compounds as part of the
Molecular Libraries Probe Production Centers Network (MLPCN) program, which
has confirmed the feasibility of this effort. The subsequent step was to generate
sufficient Artemis protein in order to screen the necessary 430,000 compounds in
the various compound libraries.
Our lab was the first to characterize the role of Artemis in opening the
hairpins in V(D)J recombination (Ma et al., 2002). Years later, we demonstrated
that Artemis is involved in nicking various DNA substrates (5’ and 3’ overhangs,
loops, flaps, and gaps) (Ma et al., 2005a). More recently, we confirmed that the 5’
exonuclease activity was indeed intrinsic to Artemis (Li et al., 2014). One
remaining question was whether Artemis had the ability to process blunt DNA
ends. If so, how does this activity fit into our structure-specific nuclease model of
Artemis?
Over the last decade, there have been ongoing efforts to create an in vitro
NHEJ reconstitution system with purified components. The vast majority of labs
utilize cellular based in vivo assays to characterize NHEJ. One major pitfall in
using a cellular system includes the inability to control for other non-NHEJ factors
17
that may complicate the interpretation of the results. In addition, cellular systems
often utilize electroporated DNA substrates that may be processed in upstream
pathways prior to reaching the site of NHEJ in the nucleus. In 2004, our lab was
the first to develop an elegant PCR-based reconstitution system that showed that
resection and polymerization occur to resolve DNA ends (Ma et al., 2004).
However, these data relied on NHEJ product detection from PCR, which does
not allow discrimination between which DNA strand is joined and has no ability to
determine the efficiency of NHEJ events. The development of an in vitro
reconstitution system will allow us to compare the NHEJ efficiencies and
determine the affects of the more recently discovered NHEJ factors, XLF and
PAXX.

18
CHAPTER 2.
EXPERIMENTAL DESIGN AND PREPARATION

2.1 OLIGONUCLEOTIDES
The Oligonucleotides used in this study were synthesized by Integrated DNA
Technologies, Inc. (San Diego, CA). The oligonucleotides were purified using
denaturing PAGE, and the concentrations were determined
spectrophotometrically. 5’ end labeling of DNA substrates was done with [γ-
32
P]
ATP (3000 Ci/mmol) (PerkinElmer Life Sciences) and T4 polynucleotide kinase
(PNK) (New England Biolabs) according to the instructions of the manufacturer.
Unincorporated radioisotope was removed by using Sephadex G-25 spin
columns (Enzymax). To create dsDNA, the unlabeled complementary strand was
added, and the sample was boiled for 5 min to inactivate T4 PNK. The DNA was
allowed to slowly cool to room temperature and then incubated overnight at 4 °C
to promote proper base pairing of the complementary strand.
3’ end labeling was performed by fill-in synthesis of [α-
32
P] dNTP (3000
Ci/mmol) (PerkinElmer Life Sciences) onto pre-annealed dsDNA with a 1-nt 5’
overhang with the Klenow fragment of DNA polymerase I (3’ -> 5’ exo
-
) (New
England Biolabs) to create a blunt-ended substrate. Unincorporated radioisotope
was removed as stated above.
The sequences of the oligonucleotides used in this study were as follows:
HC33, 5’ -GCG GAG TGT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA
19
CGT GCT AGA CTA CTG GTC AAG CGG ATC TTA GGG G- 3’ ; HC34, 5’ -CCC
CTA AGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG ATT GCA TCC
GTC AAG TAA GAT GCA GAC ACT CCG C- 3’ ; HC57, 5’ -TTT TAG TGT CTG
CAT CTT ACT TGA CGG ATG TTT T- 3’ ; HC58, 5’ -AAA ACA TCC GTC AAG
TAA GAT GCA GAC ACT AAA A- 3’ ; HC73, 5’ -phosphate-AAA ACA TCC GTC
AAG TAA GAT GCA GAC ACT AAA A-biotin- 3’ ; HC76, 5’ -biotin-AAA ACA TCC
GTC AAG TAA GAT GCA GAC ACT AAA A- 3’ ; HC77, 5’ -phosphate-A*A*A*
A*C*A* T*C*C* G*TC AAG TAA GAT GCA GAC ACT AAA A-biotin- 3’ ; HC79, 5’
-AAA AAG TGT CTG CAT CTT ACT TGA CGG ATG TTT T- 3’ ; HC80, 5’ -GGG
GAG TGT CTG CAT CTT ACT TGA CGG ATG TTT T- 3’ ; HC82, 5’ -biotin-AAA
ACA TCC GTC AAG TAA GAT GCA GAC ACT TTT T- 3’ ; HC83, 5’ -biotin-AAA
ACA TCC GTC AAG TAA GAT GCA GAC ACT CCC C- 3’ ; HC84, 5’ -biotin-AAA
ACA TCC GTC AAG TAA GAT GCA GAC ACT GGG G- 3’ ; HC85, 5’ -TTT TTT
TTT TTT TAG TGT CTG CAT CTT ACT TGA CGG ATG TTT T- 3’ ; HC86, 5’ -
TTT TAG TGT CTG CAT CTT ACT TGA CGG ATG AAA- 3’ ; HC87, 5’ -TTT
TAG TGT CTG CAT CTT ACT TGA CGG ATG GGG- 3’ ; HC89, 5’ -phosphate-
TTT TCA TCC GTC AAG TAA GAT GCA GAC ACT AAA A-biotin- 3’ ; HC90, 5’ -
phosphate-CCC CCA TCC GTC AAG TAA GAT GCA GAC ACT AAA A-biotin- 3’
; HC98, 5’ -CCC CCA TCC GTC AAG TAA GAT GCA GAC ACT AAA A-biotin- 3’
; HC99, 5’ -GGC CAG TGT CTG CAT CTT ACT TGA CGG ATG TTT T- 3’ ;
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
20
GTG ACA GGA TCC TCC ATC AAG TAA GAT GCA GAT ACT TAA CG -Biotin-
3’ ; HC 105, 5’- CTA GAC TAC TGG TCA AGC -3’ ; HC114, 5’- TGT ACA TAT
ATC AGT GTC TGC -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’ ; HC116, 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 CGG GG -3’ ; HC119, 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 CTT TT -3’ ;
HC120, 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 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’ ; HC123, 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 CGG -3’ ; HC124, 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 CG -3’ ; JG187, 5’- TGC TAG ACT ACT GGT
CAA GC -3’; JG188, 5’- TGC ATC CGT CAA GTA AGA TG -3’ ; JG226, 5’- ACG
AGC CCG ATC CGC TTG ACC AGT AGT CTA GCA CGT GAC GAT TGC ATC
CGT CAA GTA AGA TGC AGA TAC TTA AC -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 -ddG-3’. (The asterisks represent
21
phosphorothioate bonds and ddG represents a dideoxyguanidine)

2.2 PROTEIN EXPRESSION AND PURIFICATION
Wild-type Artemis and catalytically inactive Artemis
H115A
(ARM14) were
purified as described previously (Pannicke et al., 2004) (Li et al., 2014). Briefly,
Sf9 insect cells (Life Technologies, catalog no. 11496-015) were infected with
baculovirus containing C-terminal His-tagged Artemis DNA. Cells were lysed and
purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, anion
exchange chromatography, and size exclusion chromatography. The Ku70-Ku80
complex was purified as described previously (Ma et al., 2002). Briefly, Hi-5
insect cells (Invitrogen, catalog no. B855-02) were co-infected with baculoviruses
containing the DNA for C-terminal His-tagged Ku70 and untagged Ku80. Cells
were lysed and purified by nickel-nitrilotriacetic acid affinity chromatography,
dsDNA (oligo) affinity chromatography, and anion exchange chromatography.
The purification of endogenous DNA-PKcs from HeLa cells has been described
previously (Goodarzi and Lees-Miller, 2004). Ku and DNA-PKcs were confirmed
to have no detectable nuclease activities. At least three entirely independent
fresh preparations of Artemis and DNA-PKcs were used in the subsequent
assays, and these preparations generated indistinguishable results from one
another. Soluble XLF-myc-his protein was expressed in 293T cells and purified
by Ni-NTA and anion exchange chromatography. Native Pol µ and Pol λ were
purified as described previously (Gu et al., 2007a). XLF-myc-his protein was
purified from 293T cells by Ni-NTA affinity and anion exchange chromatography
22
as described (Lu et al., 2007). PAXX was purified from a pHAT4 vector encoding
for his-PAXX (gift from Dr. Stephen P. Jackson) in BL21(DE3) E. coli using a Ni-
NTA affinity chromatography, followed by TEV protease-mediated cleavage of
the N-terminal his-tag, and size exclusion chromatography as described (Ochi et
al., 2015).

2.3 ARTEMIS-HIS PURIFICATION FOR HTS DRUG SCREEN
Codon optimized baculovirus encoding for Artemis-his was amplified by
infecting Sf21 cells at MOI = 0.1. Cells were grown in Hink’s TNM-FH (Cellgro,
Catalog # 13-100-CV) supplemented with 10% fetal calf serum and penicillin-
streptomycin in 100 x 20 mm tissue culture dishes (Cellstar, Catalog # 17-5166-
01). The supernatant containing baculovirus was harvested after 72 hrs at 27 °C.
The supernatant was used to infect Sf9 insect cells at MOI = 2. Cells were grown
in SF900 II SFM (Invitrogen, Catalog # 10902-104) in 490 cm
2
roller bottles
(VWR, Catalog # 62404-370) at 130 RPM in a 27 °C incubator for 72 hrs. The
harvested cell pellet was lysed by sonication (Branson Sonifier 450) in 50 mM
NaH
2
PO
4
(pH 7.8), 500 mM KCl, 2 mM β–mercaptoethanol, 10% glycerol, 0.1%
triton X-100, and 20 mM imidazole (pH 7.8) supplemented with protease
inhibitors (PSMF, Leupeptin, Aprotinin, and Pepstatin A). The clarified extract
was batch bound to 2 mL Ni-NTA resin/ 100 mL cell culture for 2 hrs at 4 °C. The
matrix was then washed with buffer containing 50 mM imidazole (pH 7.8).
Artemis-his was eluted with 300 mM KCl and 250 mM imidazole (pH 7.8)
containing buffer. The eluates were diluted 3-fold to bring down the [KCl] to
23
approximately 100 mM. Sample was applied to a Mono Q 5/50 GL column in 50
mM Tris-Cl (pH 7.5), 10% glycerol, 1 mM DTT, 2 mM EDTA, 100 mM KCl, and
0.1% triton X-100 containing buffer. Artemis-his was fractionated using a gradient
of 100 mM to 1 M KCl. Artemis-his eluted at approximately 250 mM KCl. Artemis-
his containing fractions were pooled, dialyzed into 25 mM Tris-Cl (pH 7.5), 100
mM KCl, 10% glycerol, and 1 mM DTT containing storage buffer and flash frozen
for storage at -80 °C.

2.4 BIOCHEMICAL NUCLEASE ASSAY
The in vitro DNA nuclease assays were performed in a volume of 10 µl
with a buffer composition of 25 mM Tris-HCl (pH 8.0), 75 mM KCl, 10 mM MgCl
2
,
and 1 mM DTT. The reactions consisted of 20 nM of
32
P-labeled DNA incubated
with 0.5 mM ATP (or 0.5 mM AMP-PNP), 50 nM Ku, 50 nM DNA-PKcs, and 50
nM Artemis (or 50 nM ARM14) at 37 °C for 30 min unless specified otherwise.
The time course assays were performed in a volume of 12 or 15 µl. The ladders
were created by incubating 40 nM
32
P-labeled ssDNA with 0.5 milliunits/ml snake
venom phosphodiesterase I (Sigma-Aldrich, catalog no. P3243) for 15 min at 37
°C. All reactions were stopped by the addition of an equal volume of 98%
formamide, heated to 95 °C for 5 min, placed on ice for 5 min, and analyzed on a
12 or 14% denaturing PAGE. The gels were then dried and exposed to a
phosphorimaging screen overnight. The screen was scanned, and quantification
was performed in Quantity One
®
(Bio-Rad).

24
2.5 RECONSITUTION ASSAY
In vitro NHEJ assays were performed in a volume of 10 µl with a buffer
composition of 25 mM Tris-HCl (pH 8.0), 75 mM KCl, 10 mM MgCl2, and 1 mM
DTT, 10% PEG (8,000). The reactions consisted of 20 nM of 32P-labeled DNA
and 20 nM of unlabeled DNA substrate incubated with 0.5 mM ATP and 200 nM
streptavidin (in reactions with biotinylated substrates) at 37°C for 60 minutes,
unless specified otherwise. In addition, 100 uM dNTPs were added to reactions
containing a polymerase. The streptavidin is used to block the biotinylated-end of
the DNA from proteins. Phenol-chloroform extraction was immediately performed
on the reactions to de-proteinize the samples. Reactions were then analyzed on
an 8% denaturing PAGE. The gels were then dried, exposed to a
phosphorimager screen overnight. The screen was scanned and quantification
was performed in Quantity One® 1-D analysis (Bio-Rad).

2.6 JUNCTION SEQUENCE ANALYSIS
The dried denaturing PAGE gels were exposed to X-ray film for at least 24
h. The developed film was then overlaid on the gel to cut out individual NHEJ
ligation bands. The bands were incubated in 100 uL TE at 37°C overnight to
allow ample time for DNA diffusion. PCR was performed on the samples using
primers (HC105 and HC114 for 5’ labeled substrates and JG187 and JG188 for
3’ labeled substrates) that flank the junction. The PCR product was then TA-
cloned into a pGEM-T Easy vector by following the product manual (Catalog #
TM042, Promega). TA-cloned product was diluted 2-fold in ddH2O and
25
transformed into DH10B competent cells and transformants were plated on
LB/Amp/X-gal agar plates. White colonies were selected for plasmid mini-prep
and Sanger sequencing (Genewiz).

26
CHAPTER 3.
HIGH THROUGHPUT SCREEN FOR INHIBITORS OF THE
ARTEMIS ENDONCULEASE FOR CANCER THERAPY:
OPTIMIZATION AND SCALING OF ARTEMIS
PURIFICATION FROM INSECT CELLS

ABSTRACT
We had proposed to initiate a high throughput screen (HTS) for inhibitors of
Artemis, the human nuclease involved in V(D)J recombination, for the treatment
of acute lymphoblastic leukemia (ALL). ALL accounts for almost 30% of pediatric
cancers. Although survival has improved with conventional chemotherapeutic
approaches, novel treatments are required to further improve survival and
decrease the occurrence of relapses while reducing adverse affects. The
discovery of an Artemis inhibitor would be a novel first-in-class nuclease inhibitor.
This project initially used a Molecular Libraries Probe Production Centers
Network (MLPCN) funded screen of over 30,000 compounds to confirm that the
Artemis biochemical assay was compatible with the 1,536 well-plate format to
screen for inhibitors. A large amount of Artemis was required in order to screen
>430,000 compounds with the intention of identifying an Artemis inhibitor. The
existing Artemis purification protocol needed to be optimized for large-scale
purification for the screening efforts. We optimized purification by codon
optimization for increased expression, increased the amount of Ni
2+
-affinity resin
27
to increase yield, and used fine-resin anion-exchange chromatography to
increase specific activity. These modifications have allowed us to scale Artemis
purification to produce 8 mg per 1 L cell culture.

INTRODUCTION
Acute lymphoblastic leukemia is a blood cancer that Affects children and
adults but is most common in patients between 2 and 5 years of age. In 2016, an
estimated 6,500 new cases will be diagnosed (Siegel et al., 2016). Survival in
childhood ALL is approaching 90% but novel treatments are required for infants,
adults, and stubborn relapse patients (Inaba et al., 2013). While the factors that
trigger ALL is not known, it is thought that a combination of exogenous
exposures to carcinogens and endogenous genetic factors may cause the
genetic rearrangements that stimulate the uncontrolled growth of B- or T-
lymphocytes.
ALL cells of most patients express RAG-1 and -2 proteins which are
required in V(D)J recombination to produce the DNA hairpin intermediates in the
V, D, and J gene segments. Artemis is then required to open the DNA hairpin to
join the gene segments. If the hairpin is not opened, a deleterious chromosomal
break arises. Artemis is the only enzyme in vertebrates that can open these
hairpin structures efficiently. Thus, a specific Artemis inhibitor would be useful as
a selective target for ALL cells since they are undergoing V(D)J recombination
(Figure 3.1A). A potential drug would be highly selective for ALL tumor cells, with
only a subclinical toxicity to the new waves of pre-B and –T lymphocytes.
28
We have developed a biochemical fluorescent-based assay utilizing
Artemis endonuclease and hairpin opening activity in Mn
2+
containing buffers. It
is thought that the Mn
2+
is able to interact with Artemis to change its
conformation, whereas DNA-PKcs and Mg
2+
are required physiologically. The
substrate for the HTS contains a 12-bp perfect hairpin with an 8-nt 5’ dT
overhang. The first four 5’ nucleotides have phosphorothioate bonds to inhibit the
activity of potential contaminating exonucleases. The 5’ end contains a 6-
fluorescein amidite (6-FAM) moiety with an absorbance maximum of 492 nm and
emission maximum of 517 nm, and the 3’ end contains a black hole quencher to
block background fluorescence (Figure 3.1B). Sanford Burnham Prebys (SBP)
Medical Discovery Institute (San Diego, CA) successfully used 125 nM of this
substrate with 30 nM Artemis to test over 30,000 compounds at 20 uM in a 1,536
well plate format for the initial MLPCN screen. This screen yielded 235
compounds that produced a 30% inhibition, in which only 153 were subsequently
re-confirmed in triplicate. The success of this initial screen justified a larger HTS,
and the purification of large amounts of Artemis for the screen of >430,000
compounds.
29

FIGURE 3.1. HTS assay is designed to find inhibitors of V(D)J recombination. A, V(D)J
recombination creates a DNA hairpin intermediate that must be opened by the Artemis:DNA-
PKcs complex. B, The biochemical assay for the HTS utilizes a hairpin substrate with a 5’ 6-FAM
and 3’ black hole quencher. Artemis endonuclease activity will cut off the 5’ overhang nick the
DNA hairpin. The overhang cutting will serve as a proxy for Artemis activity. The first four
nucleotides have phosphorothioate bonds to inhibit cutting from potential contaminant
exonucleases.

Baculoviral Expression System for Artemis Purification
The recombinant Artemis protein we had originally attempted to purify for
the HTS was a C-terminally his-tagged variant expressed in Spodoptera
frugiperdia 9 cells (Sf9). This is an insect cell that has been adapted to grow in
the absence of serum, and can be utilized in a baculoviral expression system for
protein purification. In the baculoviral expression system, the polyhedrin gene is
replaced with the recombinant protein of interest. The baculovirus has the ability
to infect insect cells and exhibit both lytic and occluded life cycles. In the early
phase, the viral DNA is replicated. Next, during the late phase, the viral genes
are expressed, and the resulting baculoviruses bud to form extracellular viruses.
The supernatant can be collected in order to amplify and collect baculoviruses for
subsequent infections. The very late phase consists of expression of the
30
polyhedrin or recombinant protein and cell lysis starts to occur with production of
occluded viruses. It is essential to collect and use extracellular viruses and avoid
occluded viruses to maintain high viral titers to maximize protein yield.
Past experience in our lab has shown that E. coli purified Artemis is not active,
suggesting that post-translational modifications and/or eukaryotic protein folding
processes are required to create the active enzyme. Furthermore, Artemis
purified from human embryonic kidney (HEK) 293 cells contains contaminating
exonucleases that may complicate data interpretation. Thus, the protocol called
for infecting Sf9 cells with a baculovirus encoding for C-terminally his-tagged
Artemis. The cells were then lysed and the clarified supernatant was applied to
Ni
2+
resin (Ni-NTA) to bind the his-tagged Artemis protein. The eluate was then
applied to an anion exchange resin that contains a diethylaminoethyl (DEAE)
functional group in a buffer containing 300 mM NaCl. At this salt concentration,
Artemis-his does not bind to the resin and is separated from the more negatively
charged molecules that remain bound. The rationale behind this two-step method
was to maximize yield and minimize the time. This method proved to be
inadequate in providing high quality enzyme for the HTS. In addition, a contract
research organization (CRO) attempted to purify Artemis-his using an alternative
two-step method utilizing Ni
2+
affinity followed by size exclusion chromatography
(SEC) using fast protein liquid chromatography (FPLC), which resulted in inactive
enzyme.

31
RESULTS
Codon Optimization Increases the Yield of Artemis purified
Southern Research Institute (Birmingham, AL) created and provided us
baculovirus encoding for an insect cell codon-optimized carboxy-terminal 6x
histidine tagged Artemis. To assess the quality of expression from our
baculovirus, we infected adherent Sf21 cells using 0.1, 0.5, and 1% baculoviral
supernatant and separated the proteins in the whole cell lysate (WCL) using
denaturing PAGE after 72 hrs of infection (Figure 3.3A). The expression of
Artemis-his from the codon optimized baculovirus is slightly improved compared
to non-codon optimized baculovirus (Figure 3.3A, lane 2 vs. 3). Next, we
performed a small-scale expression test in Sf9 cells to determine the time at
which the protein expression is maximal. Cells were collected 0, 24, 48, 72, and
96 hrs post-infection and the WCL was applied to an SDS-PAGE gel. Indeed,
Artemis-his expression is maximal at 72 hrs with 73% cell viability as determined
by trypan blue exclusion (Figure 3.2B). Therefore, we had decided to harvest Sf9
cells at 72 hrs post-infection for maximal Artemis-his yield.
32

FIGURE 3.2. Codon optimized Artemis baculovirus can be expressed in insect cells. A,
Sf21 insect cells were infected with 0.1% (v/v) non-codon optimized baculoviral supernatant (lane
2) or codon optimized baculovirus in increasing concentrations (lanes 3-5). Cells were harvested
after 72 hrs and WCL was applied to denaturing PAGE. B, Codon optimize baculovirus was used
to infect Sf9 cells for Artemis-his expression test to determine the optimal time to harvest cells.
Cell viability data was obtained using trypan blue exclusion on cells prior to lysis.

Artemis-his purified in Denaturing Conditions is Not Active
We had a suspicion that Artemis-his had a tendency to aggregate from our
experiences with this protein (Figure 3.3). Firstly, in Ni
2+
affinity chromatography,
the force of gravity was not strong enough to allow the flow of buffer, indicating
that aggregation or precipitation could be occurring on the column. Secondly, the
CRO, using size exclusion chromatography, indicated that their column’s
backpressure had increased after multiple rounds of purification, which further
indicates that protein is precipitating. Finally, the Artemis-his purified utilizing our
Ni-NTA and batch DEAE two-step method had a tendency to precipitate in the
HTS 1536-well format assay.
33

FIGURE 3.3. Artemis-his purification scheme. Purification of Artemis-his was initially attempted
via (1) Ni-NTA followed by size exclusion chromatography by a CRO and (2) Ni-NTA followed by
batch anion exchange chromatography. All steps of purification has indicated larger aggregates
that may lead to precipitated or inactive enzyme (red box).

The potential precipitation occurring during Ni
2+
affinity chromatography
led us to attempt purification in denaturing conditions. The use of a strong
chaotropic agent in guanidinium hydrochloride (Gu-HCl) is able to destabilize the
secondary structure of proteins by disrupting its interaction with water molecules.
The linearized enzyme will then have an exposed his-tag that may initially be
occluded in its native state. After binding, the matrix is washed successively with
decreasing Urea concentrations to promote re-folding of the his-tagged protein
(Figure 3.4A). We lysed insect cells expressing Artemis-his in buffer containing 6
M Gu-HCl and applied the clarified supernatant to the Ni-NTA matrix. The matrix
was then washed with 6 M Urea, 3 M Urea, 1.5 M Urea, 0.75 M Urea, and then
eluted in buffer without Urea (Figure 3.4A). Increasing amounts of Artemis-his is
found in the washes with decreasing Urea concentrations, suggesting that re-
folding may be favoring the dissociation of Artemis-his from the Ni-NTA matrix
34
(Figure 3.4B). Nevertheless, Artemis-his was eluted with 250 mM imidazole
(Figure 3.4B, lane 6). This fraction was tested for Artemis hairpin-opening and
endonuclease activity in a gel-based assay using 5’-radiolabeled DNA hairpin
substrate with an 8-nt 5’-overhang (SL23). The first four nucleotides contain
phosphorothioate bonds to suppress processing from contaminating
exonucleases and thus activity can be attributed to Artemis-his. The H1 Artemis
prep and Hi-Trap Q fractions were used as positive controls and were subjected
to increasing concentrations of putative Artemis inhibitor in 10% DMSO (Figure
3.4C, lanes 2 to 21). Artemis-his purified under denaturing conditions was not
active (Figure 3.4C, lanes 22 to 26). Previous purifications have demonstrated
that Artemis-his eluted from the Ni-NTA matrix is active (data not shown). Thus,
Artemis-his purified in denaturing conditions results in inactive protein, which is
most likely due to Artemis being misfolded.
35

FIGURE 3.4. Artemis-his purified in denaturing conditions is not active. A, Workflow of
Artemis-his purification in denaturing conditions and on-column re-folding. B, SDS-PAGE of
samples from Urea wash steps and Ni-elution. C, Artemis activity assay using 60 nM Artemis-his
and increasing amounts of possible Artemis inhibitor for 30 min at 37 ºC. Artemis-his H1 prep was
purified using Ni-NTA in native conditions and Mono Q FPLC (lanes 2-6). F21 (lanes 7-11), F22
(lanes 12-16), and F23 (lanes 17-21) are fractions from Artemis-his purified using Ni-NTA in
native conditions and Hi-Trap Q. Gu-HCl (lanes 22-26) is the Ni-eluate from Artemis-his purified
in denaturing conditions.

Increasing Ni-NTA Resin Increases the Yield of Artemis Purified
In order to purify large quantities of Artemis-his in insect cells, we sought
to examine whether the amount of Ni-NTA used currently was sufficient to bind
all soluble Artemis-his. The published technical manual from QIAGEN state that
the binding capacity for their Ni-NTA resin is 50 mg his-tagged protein per mL of
resin (QIAGEN, 2008). We had been using 100 ul of Ni-NTA for 100 mL of cells,
which is sufficient to theoretically bind 5 mg Artemis-his. To test whether the
36
amount of Ni-NTA was limiting, we applied 19.5 ug purified Artemis-his onto 50 ul
(2.5 mg binding capacity) or 1 mL (50 mg binding capacity) of Ni-NTA resin
(Figure 3.5). The Ni-NTA flow through (Ni-FT) was collected and 1/50
th
of the FT
was loaded on to a denaturing gel. The FT from using 50 ul Ni-NTA still
contained Artemis-his whereas it was absent in the FT when using 1 mL Ni-NTA
(Figure 3.5A, lane 2 vs. B, lane 2). The subsequent washes contained no visible
Artemis-his (Figure 3.5A, lane 3 and B, lanes 3-4). Artemis-his does remain
bound after the washes (Figure 3.3A, lane 4 and B, lane 5). Even though 50 ul of
Ni-NTA is capable of binding up to 2.5 mg of his-tagged protein, it was not
sufficient to bind 19.5 ug Artemis-his (Figure 3.5A, lane 2). These data suggest
that the tertiary structure of Artemis-his may prevent the his-tag from being
sufficiently exposed to allow optimal contact with the Ni-NTA. Thus, it may be
necessary to either increase the amount of Ni-NTA used or to purify the protein
under denaturing conditions to expose the potentially occluded his-tag.

FIGURE 3.5. Increasing the amount of Ni-NTA increases Artemis-his binding. Artemis-his
(19.5 ug) was bound to 50 ul or 1 mL Ni-NTA. A, Artemis-his is bound to 50 ul Ni-NTA and flow
through (lane 2), wash (lane 3), and Ni-NTA resin post-wash (lane 4) was run on SDS-PAGE. B,
Artemis-his is bound to 1 mL Ni-NTA and flow through (lane 2), wash 1 (lane 3), wash 2 (lane 4)
and Ni-NTA resin post-wash (lane 5) was run on SDS-PAGE.
37
Artemis-his purified with increased Ni-NTA is active
The use of more Ni-NTA matrix improved yield. Thus, we sought to
confirm that the increased protein purified was indeed active. We purified
Artemis-his from 100 mL insect cells. Samples from Ni
2+
affinity chromatography
were analyzed on SDS-PAGE (Figure 3.6A). The elutions contained Artemis-his,
which migrates at approximately 100 kDa (Figure 3.6A, lanes 7 – 11). These
samples were also tested for Artemis hairpin-opening and endonuclease activity
(Figure 3.6B). There was no detectable nuclease activity in the whole cell lysate,
pellet after centrifugation of the lysate, clarified supernatant, and Ni
2+
flow-
through (Figure 3.6B, lanes 2 – 5). Ni
2+
eluates 1 – 5 show both hairpin-opening
and endonuclease activity, indicating the presence of active Artemis-his (Figure
3.6B, lanes 6 – 10).

FIGURE 3.6. Ni
2+
eluates include active Artemis-his. A, Coomassie-stained SDS-PAGE gel of
samples from Ni
2+
affinity chromatography. Lanes 7-11 contain eluates 1-5, respectively. B, Ni
2+

affinity chromatography samples are incubated with DNA hairpin substrate *SL23. Artemis hairpin
opening and endonuclease activity is observed in eluates 1-5 (lanes 6-10).

38
Non-ionic detergent does not interfere with Artemis-his activity
Aggregation could be caused by hydrophobic regions of Artemis-his that
interact to become larger complexes. One way to prevent aggregation is to utilize
denaturants in low concentrations that are sufficient to help solubilize proteins but
not completely denature them. Non-ionic detergents, such as Triton X-100 are
commonly used in protein purification to help solubilize hydrophobic proteins.
The endonuclease activity of Artemis-his is not inhibited by 0.002% to 2% Triton
X-100 and even may stimulate activity (Figure 3.7, lane 2 – 6). As expected, the
addition of 0.0001% SDS along with 0.02% Triton X-100 inhibits the activity of
Artemis-his (Figure 3.7, lane 4 vs. 7). This suggests, that non-ionic detergents up
to 2% may be able to prevent aggregation and allow more enzyme to be
available to recognize the DNA hairpin substrate. Furthermore, even a small
amount of ionic-detergent, 0.0001% SDS, most likely destabilizes Artemis-his
and/or DNA substrate to inhibit the promoting effects of Triton X-100 (Figure 3.7,
lane 7).
39

FIGURE 3.7. Triton X-100 (non-ionic detergent) does not inhibit Artemis-his activity.
Artemis activity assay using 20 nM Artemis-his on 20 nM DNA hairpin substrate for 30 min at 37
ºC. Lanes included no detergent (lane 2), 0.002% Triton X-100 (lane 3), 0.02% Triton X-100 (lane
4), 0.2% Triton X-100 (lane 5), 2% Triton X-100 (lane 6), and 0.02% Triton X-100 + 0.0001% SDS
(lane 7).

Purification in Triton X-100 containing buffers prevents the formation of
larger protein complexes
We attempted to purify Artemis-his using a three-step scheme: (1) Ni
2+

affinity chromatography, (2) anion exchange chromatography (Mono Q FPLC),
and (3) size exclusion chromatography (Superose 12 FPLC). All buffers in the
first two steps of purification contained 0.1% Triton X-100. The Mono Q fractions
were then pooled, and half the sample was either applied to a Superose 12
column in the presence of 0.1% Triton X-100 or without Triton X-100 (Figure.
40
3.8A). The chromatogram of UV
280
absorbance, which is proportional to the
amount of protein in the sample, shows that in the presence of 0.1% Triton X-
100, there is one sharp peak at fraction 31 (12.5 mL), and this peak corresponds
to Artemis-his (Figure 3.8B and D). Conversely, SEC without Triton X-100
resulted in three peaks at F20 (8.5 mL), F28 (11.5 mL), and F50 (20.5 mL), with
the majority of Artemis-his being present in the peak at F28 (Figure 3.8C and E).
The first peak (F20) consists of largely aggregated material since this peak is in
the void volume of the column, and the broad peak (F50) could be smaller
proteins or Triton X-100 that was initially in the injected sample (Figure 3.8C and
E). Using linear regression analysis, Artemis-his purified in in 0.1% Triton X-100
is approximately 150 kDa, whereas, Artemis-his purified without Triton X-100 is
approximately 310 kDa.

41

FIGURE 3.8. Triton X-100 decreases multimeric Artemis-his complexes. A, Artemis-his was
applied to SEC with or without 0.1% Triton X-100. B, UV
280
chromatogram under 0.1% Tritons X-
100 showing one sharp peak at F31. C, UV
280
chromatogram without Triton X-100 showing peaks
at F20, F28, and F28. D, Coomassie stained SDS-PAGE gel of samples from panel B shows the
majority of Artemis-his in fractions 30. E, Coomassie stained SDS-PAGE gel of samples from
panel C shows the majority of Artemis-his in fractions 26. Fraction 20 is the void volume and
fraction 50 contains no visible protein.

Artemis-his precipitates on Mono Q FPLC column
We next attempted to determine whether purifying Artemis-his using Mono
Q (anion exchange chromatography) FPLC, instead of using the DEAE matrix in
batch format, was reproducible. The latter was previously used to decrease
42
purification time and maximize protein yield but ultimately resulted in enzyme that
would precipitate in the 1536-well plate assay. We reasoned that the precipitation
was due to soluble aggregates that eventually precipitate. Thus, we had decided
to include 0.1% Triton X-100 throughout anion exchange chromatography, which
was previously determined to not effect Artemis-his activity and also help
promote deaggregation of larger protein complexes (Figures 3.7 and 3.8).
Purification of Artemis-his in 0.1% Triton X-100 consistently yielded over 7
mg Artemis-his per 1L culture with good purity and activity (Figure 3.9B). To
verify the purification results, a CRO successfully repeated the protocol with
similar purity, yield (7.3 mg/L culture), and activity (Figure 3.9C). This is in
contrast to the initial purification efforts using size exclusion chromatography that
resulted in subpar purity, yield, and activity (Figure 3.9A).

FIGURE 3.9 Artemis-his purification results are reproducible from two independent
institutions. A representative coomassie stained polyacrylamide gel with purification table with
batch ID, insect cell culture volume, protein yield, and activity (Y/N) information. A, CRO #1
purified Artemis-his via Ni
2+
-affinity and size exclusion chromatography. B, The Lieber lab purified
Artemis-his via Ni
2+
-affinity and Mono Q anion exchange chromatography. C, CRO #2 purified
Artemis-his via Ni
2+
-affinity and Mono Q anion exchange chromatography to confirm the Lieber
lab purification results.
Artemis-his
Batch Volume
(L)
Yield
(mg/L)
Ac6ve
(Y/N)
C-2 0.6 4.1 Y
NA 3.0 0.2 -
C-3 3.0 4.2 N
C-4 3.0 4.1 N
C-5 3.0 4.1 N
C-6 3.0 4.0 N
C-7 3.6 1.9 N
C-8 2.7 7.4 N
C-9 0.6 2.8 N
Batch Volume
(L)
Yield
(mg/L)
Ac6ve
(Y/N)
H16 0.1 9.9 Y
H17 0.3 7.1 Y
H18 0.3 7.4 Y
Artemis-his
Batch Volume
(L)
Yield
(mg/L)
Ac6ve
(Y/N)
R9562 0.1 7.3 Y
Artemis-his
A B C
43
We had observed increasing FPLC backpressure measurements with
successive application of post-Ni eluate to the Mono Q column, which indicates
that the sample contains material that can lead to obstruction of the column. The
Mono Q 5/50 GL column contains resin with an average particle size of 10 µm
packed between a top and bottom polyethylene filter with a 7 µm pore size
sandwiched within a porous top and bottom filter. We attempted to change the
top filter since this is the first barrier in preventing precipitated protein from being
introduced into the column. To our dismay, we noticed a film formed under the
top filter (Figure 3.10A-B). This film was then dissolved in PBS and resolved by
SDS-PAGE. Coomassie staining and anti-his western blotting shows that the
Mono Q film contained predominantly Artemis-his protein (Figure 3.10C-D). The
removal of this film reduced column backpressure from 2.4 MPa to 1.8 MPa,
suggesting that precipitated Artemis-his was creating an obstruction in the
column.

FIGURE 3.10. Precipitated Artemis-his was found under top filter of Mono Q column. A,
Diagram of Mono Q column. B, A white film was found between the top filter and the Mono Q
resin. C, Coomassie blue stained SDS-PAGE of Artemis-his (lane 1), Polymerase µ (lane 2), and
dissolved Mono Q film (lane 3). D, Anti-his western blot of Artemis-his (lane 1), Polymerase µ
(lane 2), and dissolved Mono Q film (lane 3).

44
DISCUSSION
The optimization of Artemis-his purification has led to increased yield and
activity from utilizing a codon-optimized baculovirus to increase Artemis-his
expression, increased the amount of Ni
2+
-affinity matrix to increase yield, and
utilized Mono Q anion exchange chromatography in Triton X-100 to promote the
purification of non-aggregated protein (Figure 3.11). These changes improved
the yield approximately 8-fold from 1 mg/L to 8 mg/L and increased activity 2-
fold. We suspect that the increase in activity is due to the use of Triton X-100,
which promotes the purification of deaggregated Artemis-his (Figure 3.8). These
changes allowed for us to purify Artemis-his using only 3 L insect cell culture
instead of the initial proposed 40 L. We have successfully purified and delivered
the protein to Sanford Burnham Prebys (SBP) Medical Discovery Institute to
complete the HTS of over 430,000 compounds. The hits from the screen are
being analyzed to discover a lead compound to move forward.

TABLE 3.1 Artemis-his purification summary. The initial problems, solutions, and
improvements of the changes are summarized.

45
CHAPTER 4.
UNIFYING THE DNA END PROCESSING ROLES
OF THE ARTEMIS NUCLEASE: KU-DEPENDENT
ARTEMIS RESECTION AT BLUNT DNA ENDS

ABSTRACT
Artemis is a member of the metallo-β-lactamase protein family of nucleases. It is
essential in vertebrates because, during V(D)J recombination, the RAG complex
generates hairpins when it creates the double strand breaks at V, D, and J
segments, and Artemis is required to open the hairpins so that they can be
joined. Artemis is a diverse endo- and exonuclease, and creating a unified model
for its wide range of nuclease properties has been challenging. Here we show
that Artemis resects iteratively into blunt DNA ends with an efficiency that reflects
the AT-richness of the DNA end. GC-rich ends are not cut by Artemis alone
because of a requirement for DNA end breathing (and confirmed using fixed
pseudo-Y structures). All DNA ends are cut when both the DNA-dependent
protein kinase catalytic subunit and Ku accompany Artemis but not when Ku is
omitted. These are the first biochemical data demonstrating a Ku dependence of
Artemis action on DNA ends of any configuration. The action of Artemis at blunt
DNA ends is slower than at overhangs, consistent with a requirement for a slow
DNA end-breathing step preceding the cut. The AT sequence dependence, the
46
order of strand cutting, the length of the cuts, and the Ku-dependence of Artemis
action at blunt ends can be reconciled with the other nucleolytic properties of
both Artemis and Artemis DNA-PKcs in a model incorporating DNA end
breathing of blunt ends to form transient single to double strand boundaries that
have structural similarities to hairpins and fixed 5’ and 3’ overhangs.

INTRODUCTION
Artemis is in the metallo-β-lactamase family of nucleases characterized by
conserved metallo-β-lactamase and β-CASP domains. This family of nucleases
is able to hydrolyze DNA or RNA in various configurations (Dominski, 2007).
Artemis alone has intrinsic 5’ exonuclease activity on ssDNA (Li et al., 2014). On
duplex DNA, Artemis, in complex with DNA-PKcs, has endonuclease activity on
the 5’ and 3’ DNA overhangs that are often created by pathological DNA breaks
and on DNA hairpins that are formed during V(D)J recombination. Thus, patients
lacking Artemis suffer from severe combined immunodeficiency (SCID) due to a
defect in antibody formation (Moshous et al., 2001).
We have determined biochemically that Artemis, in complex with DNA-
PKcs, resects 5’ and 3’ DNA overhangs in order to create DNA end structures
that are able to be ligated by the DNA ligase IV:XRCC4 complex (Lu et al., 2008;
Ma et al., 2002). At 5’ overhangs, Artemis cuts directly at the ss/dsDNA
boundary. However, when processing 3’ overhangs and DNA hairpins, it
preferentially leaves a 4-nt overhang (Ma et al., 2005a). Perfect DNA hairpins
have sterically-constrained tight turns, and thus have ss/ds boundaries at the last
47
2 base pairs (4-nts) (Blommers et al., 1989). This may make them structurally
similar to DNA overhangs. From these observations, we have proposed that
Artemis activity on duplex DNA can be unified under a model in which
Artemis:DNA-PKcs binds to the ss/dsDNA boundary to occupy 4-nt along the
single-stranded segment. This binding is followed by preferentially nicking on the
3’ side of those 4-nt. While the current model is extensive, it lacks information on
whether Artemis can cut blunt-ended DNA that can be generated by
chemotherapeutic agents, free radicals, or ionizing radiation (Povirk, 2012).
Thus, we explore the action of Artemis at blunt DNA ends, which appears
substantially weaker than its action at ss/dsDNA boundaries. We found that
resection at blunt end depends on the DNA sequence of the duplex end, with AT-
rich DNA ends being resected by Artemis alone but GC-ends hardly at all. When
Artemis is accompanied by DNA-PKcs, it can act at all blunt ends, but in a Ku-
dependent manner. This is the first documentation that Ku can modulate Artemis
activity. The sequence-dependence and other features of Artemis action permit
unification of all of the nucleolytic properties of Artemis into a single model for its
action at ss/dsDNA boundaries.

RESULTS
Artemis Can Resect Blunt DNA Ends But at a Much Slower Rate Than at
DNA Overhangs
In Figure 4.1, we show a summary of major Artemis substrates and
preferred positions of Artemis cutting on those substrates. The open thick arrows
48
indicate the putative orientation and position of the Artemis enzyme, and the red
arrows indicate the preferred cutting sites. Artemis is known to have
endonuclease activity on DNA hairpins and on 5’ and 3’ overhangs of duplex
DNA, which is dependent on DNA-PKcs activity (Figure 4.1, top row). Previous
biochemical data have indicated that Artemis preferentially cuts on the 3’ side of
a 4-nt ssDNA segment directly adjacent to a ss/dsDNA boundary. The other
known Artemis substrates listed in Figure 4.1 (center and bottom rows) are
physiologically relevant because they may arise from replication errors or by
inadvertent action by nuclear enzymes. Nevertheless, we found that Artemis is
able to cut these substrates on the ssDNA portion nearest the ss/dsDNA
boundaries. We subsequently wondered what would occur at blunt DNA ends
when such ends may be able breathe open to form a transient pseudo-Y
structure (Figure 4.1, center row and center column).
We initially performed a time course experiment with Artemis and DNA-
PKcs on 9-nt 5’ overhang, blunt-ended, and ssDNA substrates (Figure 4.2).
Predictably, the predominant cut on the 5’ overhang substrate is at 9 nt,
producing a blunt-ended product (Figure 4.2A, lanes 1-6). The 34-nt ssDNA
substrate (which matched the sequence of the top strand of the duplex portion of
the other substrates) produced random endonucleolytic cuts (Figure 4.2A, lanes
13-18). In addition, the apparent intense band at 18-nt may be explained by the
palindromic 5’-TGCA-3’ on the 11
th
to 14
th
-nt, allowing the substrate to self-
anneal. The 18
th
-nt is then 4-nt on the 3’ side of the substrate, resulting in a more
intense band. In contrast, the 34-bp blunt-ended substrate produced a
49
distribution of cuts 1 to 6-nt into the duplex DNA end (Figure 4.2A, lanes 7–12).
The cut products were then quantified as a percentage of the total substrate and
plotted as a function of time (Figure 4.2B). The rate of activity was determined by
calculating the slope from the average of two entirely independent experiments
(product percent per minute of reaction). (Several additional very similar
experiments further supported these observations.) The 5’ overhang substrate
was processed 3-fold faster than blunt-ended DNA and 2-fold faster than ssDNA
(Figure 4.2C). Although it is known that Artemis has endonuclease activity on
ssDNA, it was surprising to observe that the activity was only 2-fold slower than
at overhangs. Unlike homopolymeric ssDNA substrates, we suspect that the
ssDNA substrate used here may have self-annealed to produce partial duplex
DNA structures.

50

FIGURE 4.1. Optimal substrates for Artemis have ss/dsDNA boundaries that direct Artemis
to the point of preferred nuclease action. Shown are the known major substrates for Artemis
(Ma et al., 2002, 2005a). The putative orientation and position of the Artemis enzyme are
indicated by the open thick arrows, consistent with the preference of Artemis for ss/dsDNA
boundaries and its propensity to cut on the 3’ side of an 4-nt ssDNA region at this boundary. The
preferred cutting sites of Artemis are noted with red arrows. We propose that weaker activity may
be seen on variants of these structures when conditions favor DNA breathing into one of the
forms shown here. In addition to the substrates shown, Artemis has 5’ exonuclease activity at 5’
protruding DNA ends (Li et al., 2014), which is the only activity that does not rely on DNA-PKcs
(under Mg
2+
conditions) (see text). Artemis also has very weak endonuclease activity on ssDNA,
which is stimulated by DNA-PK
cs
and can be slightly stronger for ssDNA that can self-anneal or
fold back at internal positions because of self-complementarity (Gu et al., 2010).
51

FIGURE 4.2. Artemis activity comparison on blunt dsDNA and 5’ DNA overhangs. 100 nM 5’
32
P-radiolabeled substrate was incubated with 25 nM DNA-PK
cs
and 25 nM Artemis for the
indicated times at 37 °C. The substrates were a 9-nt poly(dT) 5’ overhang substrate (*HC85/58);
a 34-bp, blunt-ended substrate flanked by four dTs (*HC57/58); and a 34-nt ssDNA substrate
(*HC57). The asterisks indicate [
32
P] phosphates. A, samples from time points were run on a 14%
denaturing PAGE. B, cut products were quantified, and the average of two independent
experiments was plotted as a function of time. Error bars indicate mean S.D. (Several additional
similar time courses gave results indistinguishable from those shown here.). C, linear regression
was used to calculate the slope and R
2
values.

Artemis Endonuclease Activity on Blunt DNA Ends Is Strictly Dependent on
DNA-PKcs and ATP and is Modulated by Ku
As mentioned, Artemis has intrinsic 5’ exonuclease activity and DNA-
PKcs-dependent endonuclease activity. Therefore, we wondered whether this
activity also applied to blunt-ended DNA. We incubated a 5’-radiolabeled 34-bp
dsDNA substrate flanked with dTs in a reaction with Ku, DNA-PKcs, Artemis, and
ARM14 (catalytically inactive Artemis
H115A
) with or without ATP for 30 min at
52
37°C (Figure 4.3). Intrinsic Artemis 5’ exonuclease activity was able to resect the
first five nucleotides in the absence of DNA-PKcs and ATP (Figure 4.3; lanes 2,
4, and 7). Furthermore, Artemis 5 endonuclease activity predominately cut 1- to
6-nts from the 5’ end in the presence of DNA-PKcs (Figure 4.3, lane 7 versus 9),
and this activity was dependent on ATP (Figure 4.3, lane 4 versus lane 9).
Interestingly, Ku modulated Artemis endonuclease activity at blunt ends by
restricting the cuts to 2- and 4-nt instead of the 1- to 6-nt distribution observed in
Ku-independent reactions (Figure 4.3, lane 9 versus 10, 5’ end resection). The
addition of Ku also stimulated 3’ endonuclease activity by enabling Artemis to cut
up to 9-nt into the 3’ end (Figure 4.3, lane 9 versus 10, 3’ end resection). This 3’
activity remained DNA-PKcs-dependent (Figure 4.3, lane 8 versus 10, 3’ end
resection). Furthermore, the observed activities are not due to contaminating
nucleases because the catalytically inactive Artemis point mutant, ARM14, which
was purified in the same manner, did not show any 5’ or 3’ activity (Figure 4.3,
lanes 10-11). We also confirmed that, similar to Artemis endonuclease activity on
overhangs and ssDNA (Goodarzi et al., 2006; Gu et al., 2010), Artemis
phosphorylation is not required for its endonuclease activity on blunt-ended DNA
(data not shown), although autophosphorylated DNA-PKcs is required for all
ends.
53

FIGURE 4.3. Artemis activity on blunt dsDNA with dT ends. 20 nM 5’
32
P-radiolabeled, 34-bp
dsDNA (*HC57/58) was incubated with 50 nM Ku, 50 nM DNA-PKcs, 50 nM Artemis, 50 nM
ARM14, and 0.5 mM ATP in the respective reactions for 30 min at 37 °C, followed by resolving
the sample on a 12% denaturing PAGE. Reactions included Artemis (lane 2); Ku and Artemis
(lane 3); DNA-PKcs and Artemis (lane 4); Ku, DNA-PKcs, and Artemis (lane 5); and Ku, DNA-
PKcs, and ARM14 (lane 6). ATP was included in lanes 7-11. M, marker lane; asterisk, [
32
P]
phosphate. This is a representative gel of at least three identical experiments and at least two
preparations of Artemis and DNA-PKcs. (Several additional similar experiments gave results
indistinguishable from those shown here.)

Titration Reveals That DNA-PKcs Can Be Inhibitory at High Concentrations
Next we examined the effect of the amount of DNA-PKcs on the observed
activity of Artemis. We generated a 5’-labeled, 34-bp, blunt-ended DNA by
54
annealing 5’-labeled ssDNA to its unlabeled complementary strand containing a
5’ biotin (Figure 4.4). This substrate was then incubated along with streptavidin,
which binds biotin tightly, to prevent/inhibit Artemis action on one end of the
dsDNA to examine the activity on the labeled end only. Using a blunt-ended
substrate flanked with four dAs, we titrated the amount of DNA-PKcs in the
reaction from 2 to 105 nM (Figure 4.4). Ku and DNA-PKcs alone did not have any
detectable levels of nuclease activity (Figure 4.4, lanes 2 and 3). Artemis activity
was maximal at a 1:1 molar ratio with DNA-PKcs (Figure 4.4, lanes 6-9).
Furthermore, Artemis activity was stimulated 2- to 3-fold in the presence of
Ku (Figure 4.4, lanes 6–9 versus 10-13). Interestingly, increasing the molar ratio
of DNA-PKcs to Artemis to 2:1 reduced Artemis activity by 50% (Figure 4.4, lane
8 versus 9 and lane 12 versus 13). These data support the view that Artemis acts
in a complex with DNA-PKcs at a 1:1 molar ratio and that increasing the
concentration of DNA-PKcs may block the DNA ends from Artemis activity.
55

FIGURE 4.4. DNA-PKcs titration for Artemis activity on blunt ends. 20 nM 5’
32
P-
radiolabeled, 34-bp dsDNA (*HC79/82) with four dAs was created by annealing the radiolabeled
(asterisk) top strand with its complementary strand with a 5 biotin (B). The substrate was
incubated with 50 nM Ku, 50 nM Artemis, and various amounts of DNA-PKcs in the respective
reactions for 30 min at 37 °C, followed by resolving the sample on a 14% denaturing PAGE. In
addition, 0.5 mM ATP and 0.1 M streptavidin (SA) were included in all reactions to suppress
Artemis activity on the 3’ end. Reactions consisted of Ku (lane 2), DNA-PKcs (lane 3), Artemis
(lane 4), Ku and Artemis (lane 5), Artemis with increasing amounts of DNA-PKcs (lanes 6-9), and
Ku and Artemis with increasing amounts of DNA-PKcs (lanes 10-13). The quantified 5’ resection
percentage is noted at the bottom of the gel. This is a representative gel of at least two identical
experiments. (Several additional similar experiments gave results indistinguishable from those
shown here.)

56
The Blunt End DNA Sequence Determines the Extent to Which Ku and
DNA-PKcs Stimulate the 5’ Nuclease Action of Artemis
We then asked whether the observed Ku stimulation is sequence-
dependent at 5’ blunt ends. We generated 5’-labeled, 34-bp, blunt-ended DNA
with four dAs (Figure 4.5A) and four dGs (Figure 4.5B) by annealing 5’-labeled
ssDNA with unlabeled 5’-biotinylated complementary strands. The substrate was
then incubated with proteins as indicated, along with ATP and streptavidin.
Artemis 5’ exonuclease activity was observed with substrates that are flanked by
AT-but not GC-base pairs at the blunt DNA ends (Figure 4.3, lanes 2, 4, and 7;
Figure 4.4, lane 4; and Figure 4.5A-B, lanes 1-4). The observed exonuclease
activity was reduced when Ku was added (Figure 4.3, lane 2 versus 3, lane 4
versus 5, and lane 7 versus 8; Figure 3.4, lane 4 versus 5; and Figure 4.5A,
lanes 1-4 versus 5-8). We hypothesized that the weak base pairing in AT-rich
ends compared with GC-rich ends allowed the DNA end to breathe and form
transient ss/dsDNA boundaries to provide a substrate for Artemis.
To examine the sequence-dependent variation further, we simulated
breathing by creating a fixed 2-nt pseudo-Y structure with dA or dG unpaired
ssDNA overhangs (Figure 4.6). DNA with 5’ dGs was processed by Artemis
when forced into a pseudo-Y structure (Figure 4.6, lanes 9-12 versus 13-16).
Interestingly, Y-structure DNA with 5’ dAs was also stimulated 2- to 3-fold (Figure
4.6, lanes 1-4 versus 5-8). These data indicate that DNA breathing may allow
AT-rich blunt DNA to be cleaved by the Artemis 5’ exonuclease activity in the
absence of other proteins (DNA-PKcs and Ku).
57
In contrast, Artemis 5’ endonuclease activity is more complex. Artemis 5’
endonuclease activity was stimulated by DNA-PKcs with AT-rich blunt DNA
substrates but only mildly stimulated with GC-rich blunt DNA substrates (Figure
4.5A-B, lanes 9-12). With blunt AT ends, up to 6-nt were resected in the
presence of DNA-PKcs (Figure 4.5A, lanes 9-12). In addition, Ku stimulated the
endonuclease activity 3- to 4-fold (Figure 4.5A, lanes 9-12 versus 13-16).
Conversely, for GC ends, DNA-PKcs only slightly activated 5’ exo- and
endonuclease activities (Figure 4.5B, lanes 9-12). However, the addition of Ku
stimulated Artemis activity more than 10-fold (Figure 4.5B, lanes 9-12 versus 13-
16). In summary, Ku could modulate the activity of Artemis DNA-PKcs depending
on the AT- or GC-richness of blunt ends. For AT-rich ends, Ku stimulated Artemis
activity only 3- to 4-fold and diminished the diversity of resection; specifically, 1-
to 6-nt resection without Ku and 1- to 2-nt with Ku (Figure 4.5A, lanes 9-12
versus 13-16). In contrast, Ku stimulated Artemis activity more than 10-fold in
GC-rich ends overall, with the most frequent resection events at 1- to 2-nt (Figure
4.5B).

58

FIGURE 4.5. The Time course of 5’ endonuclease activity on blunt-ended DNA reveals that
Artemis resects up to 6-nt, requires DNA-PKcs, and is modulated by Ku to preferentially
cut 2-nt into the duplex. 20 nM 5’
32
P-radiolabeled, 34-bp biotinylated dsDNA (B) was incubated
with 50 nM Ku, 50 nM DNA-PKcs, and 50 nM Artemis for 0, 15, 30, and 60 min at 37 °C in the
lanes as indicated. In addition, 0.5 mM ATP and 0.1 M streptavidin (SA) were included in all
reactions to suppress Artemis activity on the 3’ end. The samples were resolved on a 14%
denaturing PAGE. M, marker lane; asterisk, [
32
P] phosphate. The quantified 5’ resection
percentage is noted at the bottom of the gel. This is a representative gel of at least three identical
experiments and at least two preparations of Artemis and DNA-PKcs. (Several additional similar
time courses gave results indistinguishable from those shown here.). A, 5’ dA substrate
(*HC79/82). B, 5’ dG substrate (*HC80/83).

59

FIGURE 4.6. DNA end breathing provides a substrate for Artemis 5’ exonuclease activity.
Shown is the time course of Artemis activity on blunt-ended and fixed 2-nt Y-structure DNA. 20
nM 5’
32
P-radiolabeled, 34-bp biotinylated-dsDNA (B) was incubated with 50 nM Artemis and 0.1
M streptavidin (SA) for 0, 15, 30, and 60 min at 37 °C. The samples were resolved on a 12%
denaturing PAGE. Substrates were blunt-ended dA (*HC79/82, lanes 1-4), dA Y-structure
(*HC98/76, lanes 5-8), blunt-ended dG (*HC80/83, lanes 9-12), and dG Y-structure (*HC99/84,
lanes 13-16). Asterisk, [
32
P] phosphate. The quantified 5’ resection percentage is noted at the
bottom of the gel. This is a representative gel of at least two identical experiments.
60
Artemis 3’ Endonuclease Resection Does Not Show Substantial Sequence
Dependence but Is Stimulated by DNA-PKcs and Ku
We then asked whether the sequence-dependent activity is also observed
on the 3’ end of blunt-ended DNA. 3’-labeled, 34-bp blunt-ended DNA flanked by
four dAs (Figure 4.7A) and four dGs (Figure 4.7B) was generated by fill-in
synthesis with the Klenow fragment of DNA polymerase I (3’ -> 5’ exo
-
) and [α-
32
P] dNTP. The substrate was then incubated with Ku, DNA-PKcs, and Artemis,
as indicated, in the presence of ATP and streptavidin. The unlabeled
complementary strand contained an unlabeled 5’-phosphate and a 3’-biotin to
block Artemis action on the 3’ end. 3’ exonuclease activity was observed only on
the 3’ dA DNA and slightly on 3’ dG DNA (Figure 4.7A-B, lane 2). Interestingly,
Ku did not block this 3’ exonuclease activity (Figure 4.7A-B, lane 2 versus 4).
Artemis activity on the 3’ end was only mildly stimulated by DNA-PKcs and
produced a distribution of cuts up to 5-nt into the duplex portion of the substrate
(Figure 4.7A-B, lane 2 versus 8). Ku further stimulated Artemis activity 6- to 8-
fold independent of sequence (Figure 4.7A-B, lane 8 versus 12).
Interestingly, these results are in contrast to Artemis activity observed on
the 5’ end. For one, the breathing requirement was not sequence-dependent on
the 3’ end. The mild activity stimulation by DNA-PKcs on the 3’ end, in which only
up to 8% of the substrate was cut after 60 min (Figure 4.7A-B, lane 8), was
similar to the stimulation observed on GC-rich 5’ ends (Figure 4.5B, lane 12, and
Figure 4.7B, lane 12). Secondly, the addition of Ku stimulated Artemis activity 6-
to 8-fold on the 3’ end independent of sequence (Figure 4.7A-B, lane 8 versus
61
12), whereas Artemis activity on 5’ ends was sequence-dependent (a 3- to 4-fold
increase for AT-rich and a more than 10-fold increase for GC-rich).

FIGURE 4.7. The Time course of 3’ endonuclease activity on blunt-ended DNA reveals that
Artemis resects predominantly 1- to 3-nt, requires DNA-PKcs, and is stimulated by Ku. 20
nM 3’
32
P-radiolabeled, 34-bp biotinylated dsDNA (B) was incubated with 50 nM Ku, 50 nM DNA-
PKcs, and 50 nM Artemis for 0, 15, 30, and 60 min at 37 °C in the lanes as indicated. In addition,
0.5 mM ATP and 0.1 M streptavidin (SA) were included in all reactions to suppress Artemis
activity on the 5’ end. The P on the bottom strand denotes a cold phosphate group on the 5’ end.
The samples were resolved on a 14% denaturing PAGE. M, marker lane; asterisk, [
32
P]
phosphate. The quantified 3’ resection percentage is noted at the bottom of the gel. This is a
representative gel of at least three identical experiments and at least two preparations of Artemis
and DNA-PKcs. (Several additional similar time courses gave results indistinguishable from those
shown here.). A, 3’ dA substrate (HC86*/89). B, 3’ dG substrate (HC87*/90).

5’ Resection Occurs Prior to 3’ Resection on Blunt DNA Ends
We determined that Artemis performs asymmetric resection for the 5’
versus 3’ strands. Resection on the 5’ strand at AT-rich blunt ends occurred in a
largely Ku-independent manner. GC-rich 5’ ends were mildly stimulated by DNA-
PKcs and stimulated strongly by Ku. Conversely, Artemis exhibited sequence-
A B
B SA
3’ end
resec*on 2nt
1nt
4nt
DNA-PKcs
Artemis
Time (min) 0
M
+
15
10
+
+ + +
11
+
30
+
12
+
60
9
Ku + + + +
0
+
15
6
+
+ + +
7
+
30
+
8
+
60
5
- - - -
-
4
+
60
+
60
+
60
3
-
- +
2
- -
60
-
-
1
-
substrate
3nt
5nt
0%
1%
0%
0%
2%
0%
1%
2%
6%
2 5 3 4 1 9 6 7 8
3’ resec5on %
4%
13%
45%
10 11 12
* AAAA
34 bp
3’ end
resec*on
2nt
1nt
4nt
DNA-PKcs
Artemis
Time (min) 0
M
+
15
10
+
+ + +
11
+
30
+
12
+
60
9
Ku + + + +
0
+
15
6
+
+ + +
7
+
30
+
8
+
60
5
- - - -
-
4
+
60
+
60
+
60
3
-
- +
2
- -
60
-
-
1
-
substrate
3nt
5nt
0%
1%
0%
0%
1%
0%
1%
3%
8%
2 5 3 4 1 9 6 7 8
3’ resec3on %
12%
35%
51%
10 11 12
B SA
* GGGG
34 bp
62
independent activity on 3’ ends that required Ku for robust activity. Because it
appeared that 5’ end resection was more efficient, we asked whether 5’ end
processing was required prior to 3’ end processing. We annealed a 5’-labeled,
34-nt oligo to its 3’-biotinylated complementary oligo with or without 10
phosphorothioate bonds on the 5’ end. A phosphorothioate bond, formed by the
sulfurization of a non-bridging oxygen in the phosphodiester bond, suppresses
the action of nucleases. We performed a time course of Artemis activity in the
presence of DNA-PKcs and Ku. We observed that Artemis activity on the 3’ end
of dsDNA substrate was reduced significantly when the complementary strand
was unable to be cut first (Figure 4.8A, lanes 1-5 versus 6-10). Therefore, it
appears that the 5’ strand resection occurs first, always leaving a 3’ ssDNA
overhang that, by its ssDNA nature, does not have any breathing requirement.
This allows the 3’ overhang resection to be sequence-independent.

63

FIGURE 4.8. Artemis 5’ end resection occurs before 3’ end resection and progresses
internally along the DNA with time. Shown is the time course of Artemis action. A, 20 nM 5’
32
P-radiolabeled, 34-bp dsDNA with four dTs on the 3’ end was created by annealing the
radiolabeled (asterisk) top strand with its complementary strand with a cold 5’ phosphate (P) and
3’ biotin (B) (*HC57/73, lanes 1-5) or its complementary strand with 10 phosphorothioate bonds
on the 5’ end (*HC57/77, lanes 6-10). The substrates were incubated with 50 nM Ku, 50 nM DNA-
PKcs, and 50 nM Artemis for 0, 15, 30, 60, and 120 min at 37 °C and analyzed on a 12%
denaturing PAGE. This is a representative gel of at least three identical experiments. (Several
additional similar time courses gave results indistinguishable from those shown here.). B, 20 nM
5’
32
P-radiolabeled, 73-bp dsDNA (*HC33/34) was incubated with 50 nM Ku, 50 nM DNA-PKcs,
50 nM Artemis, and 0.5 mM ATP for 0, 10, 20, 40, 80, and 160 min at 37 °C and analyzed on a
12% denaturing PAGE. M, marker lane. This is a representative gel of at least 10 experiments
using blunt-ended DNAs of various lengths.

64
Artemis Resection Progresses Internally along the DNA with Time
Because we established that Artemis first acts on the 5’ end prior to acting
on the resulting 3’ overhang, we wondered whether this process would repeat
until the DNA was processed completely. We initially showed that a 34-bp, blunt-
ended DNA could be processed up to 11-nt after 2 h (Figure 4.8A, lane 5), which
would leave 23-bp remaining. We then incubated a 73-bp DNA with Ku, DNA-
PKcs, and Artemis for up to 160 min (Figure 4.8B, lanes 1-6). The resulting gel
showed that Artemis endonucleolytically resected up to 4-nt on the 5’ end (Figure
4.8B, lane 6, 5’ end resection). Processing on the 3’ end proceeded until 25- to
30-nt remained (Figure 4.8B, lane 6, 3’ end resection). Because we know that
26-bp are required for maximal Ku-dependent DNA-PKcs activity (West et al.,
1998), it is likely that Artemis resection proceeds until the DNA is long enough for
one Ku DNA-PKcs complex to remain bound to the DNA.

DISCUSSION
Under physiological conditions (Artemis plus DNA-PKcs in Mg
2+
solutions),
we found that resection at blunt DNA ends depends on the DNA sequence.
However, when Ku is also present, the disparity is eliminated, and blunt ends of
all sequences are resected efficiently. This is the first biochemical documentation
showing that Ku can affect the nucleolytic behavior of Artemis. Further study of it
has revealed major unifying features of the Artemis nuclease.

65
Unifying Kinetic Features for Artemis Action at DNA Ends
Previous work on Artemis has documented its action at ss/ds- DNA
boundaries, specifically at hairpins and overhangs and gaps and flaps (Ma et al.,
2002, 2005a). One study also described endonuclease action by Artemis alone in
Mg
2+
solutions at blunt DNA ends (Yannone et al., 2008). But this latter finding
was curious because no other laboratory has found endonuclease activity of
Artemis alone under Mg
2+
conditions. We noted that numerous freeze and thaw
cycles of stored Artemis enzyme preparations eventually cause such behavior,
suggesting a denaturation-induced change in the enzyme that partially eliminates
the requirement for DNA-PKcs. In addition, Artemis alone can cut overhangs in
Mn
2+
solutions, suggesting a permanent conformational change of the enzyme in
the presence of this divalent cation (Gu et al., 2010; Huang et al., 2009; Li et al.,
2014). For this reason, evaluation of Artemis under conditions where it is
dependent on DNA-PKcs was necessary to consider its function under
physiologic conditions at blunt DNA ends and to integrate this into the spectrum
of Artemis nuclease activities.
Our finding of resection by Artemis alone in Mg
2+
solutions at AT-rich DNA
ends but not at GC-rich DNA ends suggested that DNA end breathing is required
for Artemis action. In other studies, we have documented the increased breathing
of AT- rich DNA ends compared with GC-rich ends (Tsai et al., 2009). When we
“forced” breathing here by creating a fixed, 2-nt Y-structure in which the 2-nt
arms of the Y cannot anneal, we found that, indeed, the DNA sequence of the
arms no longer matters because the ends are in a permanent ssDNA
66
configuration.
A requirement for end breathing for Artemis to act on a blunt-end explains
why such end resection by Artemis is slower than overhang resection. The
breathing step is required, and the end is only in the available configuration
(ss/dsDNA) for cutting for a fraction of the time.

A Unifying Physical Model for Artemis Nuclease Activity at DNA Ends
On the basis of the findings here and shown previously, it is possible to
formulate a model that encompasses all of the nuclease activities of Artemis.
These include hairpin opening (2-nt 3’ of the tip), 5’ overhang cutting (primarily at
the ss/dsDNA boundary), 3’ overhang cutting (primarily 4-nt 3’ of the ss/dsDNA
boundary), blunt-end resection (slower than ss/dsDNA boundary substrates), and
cutting at all DNA structures containing ss/dsDNA boundaries (gaps, flaps,
heterologous loops, and Y-structures). We note that poly(dT) ssDNA can be cut
by Artemis DNA-PKcs but that this cutting is much slower than for DNA
containing a ss/dsDNA boundary (Gu et al., 2010). The apparently efficient
cutting of ssDNA shown in Figure 4.2B is due to this ssDNA being a mixed
sequence (not poly(dT)) and being capable of forming secondary structures.
For optimal activity, we have proposed previously that the Artemis enzyme
recognizes ss/dsDNA boundaries and cuts on the 3’ side of a 4-nt ssDNA region
at this boundary. This explains the hairpin opening position, which is not at the tip
but is located 2-nt 3’ of the tip (the turn in the perfect hairpin provides the 4-nt of
ssDNA because it is largely unpaired) (Ma et al., 2002). This also explains why 5’
67
and 3’ overhangs yield different overhang products (blunt for 5’ overhangs but 4-
nt 3’ overhangs for long 3’ overhangs) (Ma et al., 2002, 2005a). DNA bending by
Artemis, and, more efficiently, by Artemis DNA-PKcs, at a 5’ or 3’ overhang may
generate a structure similar to a hairpin (Niewolik et al., 2006).
Figure 4.9 shows how these same principles apply to Artemis action at
blunt DNA ends. A blunt end can breathe into a Y-structure with short ssDNA
arms. Artemis then resects the 5’ end first. The resulting 3’ overhang is then bent
by Artemis or diffuses independently into a transient hairpin-like bend that is cut
by Artemis. Because the resulting 3’ overhang may not be sufficiently long for
efficient processing by Artemis, Ku is likely required to provide a scaffold for
Artemis DNA-PKcs to bind near the overhang.

FIGURE 4.9. DNA end breathing model for Artemis action at blunt DNA ends. The diagrams
summarize the action of Artemis at blunt DNA ends (in 10 mM MgCl
2

and 75 mM KCl). Consistent
with the optimal substrate requirements of Artemis (Figure 2.1), blunt DNA ends must first
breathe to pseudo-Y structures before efficient nuclease action can begin. Artemis first acts on
the 5’ strand of the DNA end and then on the 3’ strand (which, at that point, is a 3’ overhang). The
result of 5’ and 3’ cuts is a blunt or near-blunt end that is then subject to iterative rounds of the
same process, consistent with our results. The red arrows indicate the preferred Artemis cut sites.
The open thick arrows represent the proposed Artemis binding site and orientation on the DNA
substrates.
68
The 5’ Exonuclease of Artemis
Even the 5’ exonuclease of Artemis can be incorporated into this model.
We have noted previously that the 5’ exonuclease of Artemis is independent of
DNA-PKcs at 5’ overhangs (Ma et al., 2005a). However, for recessed 5’
overhangs or blunt ends, we find that AT-richness around such a non-protruding
5’ end is necessary for it to be cut by Artemis exonuclease activity (data not
shown), implying that the 5’ exonuclease activity of Artemis also relies on
ss/dsDNA transition structures in the substrate. Only the protruding 5’ DNA ends
are targets of the 5’ exonuclease activity of Artemis without any effect on DNA
sequence. This may arise because the protruding 5’ end can insert directly into
the active site of Artemis without any more substantial substrate positioning by
Artemis that is required for it to process all other DNA ends.

The Role of DNA-PKcs and Ku in Artemis Interaction with Its Substrate
We believe that the role of DNA-PKcs is to change the conformation of
Artemis to a form that can more easily configure DNA ends into an optimal
hairpin-like substrate. As pointed out previously, Artemis under Mn
2+
conditions
does not require DNA-PKcs for any of the reactions shown in Figure 2.1 (Gu et
al., 2010; Huang et al., 2009; Li et al., 2014).
DNA-PKcs and Ku might simply increase the length of time that Artemis
remains bound so that, at the time of DNA breathing into a transient ss/dsDNA
boundary structure, Artemis can hydrolyze the phosphodiester bond. Artemis in
vivo exists predominantly in a very salt-stable complex with DNA-PKcs, whereas
69
Ku only forms a complex with Artemis DNA-PKcs when a DNA end is present
(Lieber, 2010; Ma et al., 2002). DNA-PKcs autophosphorylation is required for
DNA-PKcs to activate Artemis endonuclease activity (Lieber, 2010). Ku improves
the affinity of DNA-PKcs alone 100-fold, from 3 x 10
-9
to 3 x 10
-11
M (West et al.,
1998), and we assume that this applies to the Artemis DNA-PKcs complex as
well. The Artemis DNA-PKcs Ku complex would be able to remain at a DNA end
longer, giving Artemis more opportunity to cut.

Biological Implications of the Unified Model of Artemis Nuclease Action for
NHEJ
The key in vivo roles of Artemis are diverse and have not been easy to
reconcile with a single definition of the substrate configuration that is optimal for
Artemis nuclease action. Among the key DNA end configurations at which
Artemis is known to act are perfect DNA hairpins during the coding joint
formation step of V(D)J recombination (Ma et al., 2002; Rooney et al., 2002,
2003), repair of 10 – 40% of ionizing radiation-induced DSBs (Riballo et al.,
2004), and repair of DSBs produced by interruption of topoisomerase II action
(Kurosawa et al., 2008). All of the structures for in vivo and in vitro action of
Artemis have ss/dsDNA boundaries (Figure 4.10). Importantly, the boundary for
hairpins is due to the imperfect base pairing at the hairpin tip (Ma et al., 2002).
However, the action of Artemis at blunt DNA ends (and in a DNA-PKcs-
independent manner) complicated this simple model (Yannone et al., 2008).
Here we confirmed the action at blunt ends, but we show that, like the other
70
endonucleolytic actions of Artemis, this too is dependent on DNA-PKcs.
Moreover, the DNA end-breathing requirement unifies this substrate
configuration with the other known in vivo and in vitro substrate configurations.

Biological Implications of the Unified Model of Artemis Nuclease Action for
V(D)J Recombination
This study is likely to explain why GC-rich coding ends suffer less end
resection than AT-rich coding ends in human pre-B cell lines (Gauss and Lieber,
1996). GC-rich coding ends will breathe less than AT-rich coding ends. This
curious but well documented finding on the sequence dependence of coding end
resection has gone without explanation for nearly two decades, but the findings
in this study provide the first explanation for how DNA end resection at coding
ends in V(D)J recombination is affected by DNA end sequence.
A second point of in vivo relevance concerns the difference in resection of
coding ends versus signal ends in V(D)J recombination. It has been unclear why
signal ends suffer end resection much less often than coding ends. Signal ends
are blunt, and they suffer end resection in wild-type human lymphoid cells but
only at 5% of all signal joints (Kulesza and Lieber, 1998). In contrast, coding
ends are opened (by Artemis DNA-PKcs) to give 3’ overhangs (usually 4-nt in
length), and these coding ends suffer end resection in over 90% of coding joints
in human pre-B cells (Gauss and Lieber, 1996). This disparity has never been
explained, but one possible explanation is raised by our finding of a slower and
weaker resection of Artemis at blunt DNA ends because of the requirement for
71
DNA end breathing described here. Such an explanation would be consistent
with the fact that end resection at signal ends decreases when Artemis is absent
(Touvrey et al., 2008) and increases when the partner of Artemis, namely DNA-
PKcs, is mutated—a point that has remained without any reasonable explanation
until now (Lieber et al., 1988).
A third point of possible in vivo relevance concerns our observation in
human cells of inverted repeats at partially resected coding ends in the final
coding joints formed in human pre-B cells (Gauss and Lieber, 1996). The Artemis
resection in 2- to 6-nt increments at blunt-ends, as in Figure 4.2, may liberate
these short oligonucleotides, allowing them to be re-ligated via the single-strand
ligation activity of the ligase IV complex (Gu et al., 2007b). This would generate
the short inverted repeats observed at partially resected coding ends (Gauss and
Lieber, 1996), which have previously been difficult to explain mechanistically until
now. Therefore, the findings here not only provide a unification of the enzymatic
activities of an important structure-specific nuclease but also integrate several in
vivo observations with this unified model.

72
CHAPTER 5.
STRUCTURE-SPECIFIC NUCLEASE ACTIVITIES
OF ARTEMIS AND THE ARTEMIS:DNA-PKCS
COMPLEX

ABSTRACT
Artemis is a vertebrate nuclease with both endo- and exonuclease activities that
acts on a wide range of nucleic acid substrates. It is the main nuclease in the
NHEJ. Not only is Artemis important for the repair of DNA DSBs in NHEJ, it is
essential in opening the DNA hairpin intermediates that are formed during V(D)J
recombination. Thus, humans with Artemis deficiencies do not have T- or B-
lymphocytes and are diagnosed with SCID. While Artemis is the only vertebrate
nuclease capable of opening DNA hairpins, it has also been found to act on other
DNA substrates that share common structural features. Here we discuss the key
structural features that all Artemis DNA substrates have in common, thus
providing a basis for understanding how this structure-specific nuclease
recognizes its DNA targets.

INTRODUCTION
Pathological DNA DSBs can be the most deleterious forms of DNA
damage. These breaks can result in cell death from the deletion of a
73
chromosomal arm or by promoting the p53-mediated apoptosis pathway
(Friedberg et al., 2006). In mammalian cells, DSBs are repaired predominantly by
the NHEJ pathway. Non-pathological DSBs are created during V(D)J
recombination and immunoglobulin heavy chain CSR, which both contribute to
the adaptive immune response. These DSBs also require the NHEJ pathway to
be resolved. Any defects in the NHEJ pathway can result in marked sensitivity to
ionizing radiation and lead to the ablation of all lymphocytes. However, NHEJ is
often imprecise, a characteristic that is useful for immune diversification in
lymphocytes, but which might also contribute to deleterious genetic alterations
that lead to cancer and perhaps aging. The Artemis nuclease plays a critical role
in NHEJ in processing various DNA end configurations at DSBs and is required
to open the DNA hairpin intermediates in V(D)J recombination. Artemis is
activated by physical contact with DNA-PKcs (Ma et al., 2002), which must be
autophosphorylated in order to stimulate Artemis activity (Goodarzi et al., 2006;
Gu et al., 2010; Ma et al., 2002).
SCID is a genetic disorder characterized by an impairment in the adaptive
immune system (Rosen et al., 1995). Humans with mutations in either Artemis or
DNA-PKcs are both diagnosed with T
-
B
-
NK
+
SCID (van der Burg et al., 2009; Li
et al., 2002). Although T
-
B
-
NK
+
SCID is rare in the general population (1 in
50,000 to 500,000 live births), there is a high incidence (1 in 2,000 live births) in
the Navajo and Apache Native Americans (Buckley et al., 1997; Jones et al.,
1991). The subset of SCID found in in these Athabascan-speaking Native
Americans is due to an autosomal recessive nonsense Artemis mutation in exon
74
8, which causes a truncation of the 692 aa protein at the 192nd aa (Li et al.,
2002). As expected these patients suffer from the early onset of serious
infections (Kwong et al., 1999). Thus, the biomedical importance of Artemis is
substantial.

UNIFYING ELEMENTS OF THE ARTEMIS SUBSTRATE
Hairpin and overhangs were initially identified as Artemis:DNA-PKcs
substrates (Ma et al., 2002). Subsequently, a wider range of DNA structures with
single- and double-strand transitions were also identified as substrates (Ma et al.,
2005a). For example, blunt-ended DNA molecules were shown to be weaker
substrates (Yannone et al., 2008). However, it was unclear what similarities and
differences distinguish these substrates.
We have now been able to develop a physical model that describes key
recognition points common to all of the known substrates of Artemis:DNA-PKcs
(Figures 5.1 and 5.2) (Chang et al., 2015). This model proposes two recognition
sites (A and B), which place the two strands within the duplex portion, directly
adjacent to the double- to single-stranded DNA boundary. A third proposed
action site (red arrowhead), which is also the catalytic site, is located 1-nt (or an
equivalent distance) on the 5’ side of recognition site B (green dot) (Figures 5.1
and 5.2); this catalytic site does not have to be on the same DNA strand as
recognition site B (see Figure 5.1, 3’ overhang substrate). In all cases,
Artemis:DNA-PKcs is able to distort the single-stranded portion of the substrate
75
into a structure resembling key features of the DNA hairpin substrate. Without
these three recognition points, Artemis:DNA-PKcs activity is negligible.

Figure 5.1. Unifying Model Describing Artemis Activity on Common Physiological DNA
Substrates. Functional data on Artemis activity suggests that it has three critical recognition
points with its DNA substrates that are structurally similar (blunt ends, 3’ -> 5’ overhangs, and
hairpin structures). Artemis may be able to distort the DNA end to create a “hairpin-like” structure
in all cases. Recognition site A (blue dot) is located on the 5’ -> 3’ (top) strand at the ss/dsDNA
boundary. Recognition site B (green dot) is located on the 3’ -> 5’ (bottom) strand directly across
from recognition site A. The proposed action site (red arrowhead) is located 1-nt (or an equivalent
distance) on the 5’ side of recognition site B (green dot), as shown in the figure. The action site
(red arrowhead) does not need to be on the same DNA strand as recognition site B, but merely
an equivalent distance and direction from recognition site B.

With these three recognition points, the cutting pattern observed for all of
the major substrates can be explained. Among the major substrates, blunt ends
are the only ones requiring a DNA end breathing step prior to action by
Artemis:DNA-PKcs (Chang et al., 2015). The requirement for the breathing step
explains some key features of the Artemis:DNA-PKcs action at blunt ends
(Chang et al., 2015). First, it explains why Artemis:DNA-PKcs acts on blunt ends
76
less efficiently than DNA termini that have an obvious double- to single-strand
boundary (Chang et al., 2015; Yannone et al., 2008). This is because the
breathed state is only very short-lived. Second, it explains why AT-rich DNA
termini are cut much faster than the more stable GC-rich DNA-termini (Chang et
al., 2015). Third, once the blunt end breathes open, the transient 5’ single-strand
is cut faster than the 3’ single-strand (Chang et al., 2015; Yannone et al., 2008).
This may be explained by steric factors, since removal of the transient 5’ single-
strand allows the 3’ single-strand to more readily assume a hairpin conformation
(Figure 5.1).
This model also explains why the resulting product of Artemis:DNA-PKcs
activity differs on 5’ and 3’ overhangs (Ma et al., 2002). The nts on a 5’ overhang
substrate are able to fold back toward the duplex to create a hairpin-like
configuration. The Artemis:DNA-PKcs complex is able to recognize this hairpin-
like structure and cut 1-nt 5’ of recognition site B, resulting in a perfectly blunt-
ended product (Figure 5.1, 5’ overhang substrate). Similarly, the nts on a 3’
overhang substrate also fold back toward the duplex to form this hairpin-like
structure. However, in this scenario, the 4
th
nt of this overhang is spatially located
a distance equivalent to 1-nt 5’ of recognition site B (~3 to 5 angstroms). Thus,
Artemis:DNA-PKcs activity on this substrate results in a 4-nt overhang at the 3’
end (Figure 5.1, 3’ overhang substrate) (Chang et al., 2015). This model
requires a polarity in the Artemis:DNA-PKcs complex in order to recognize the
helical pitch of the double stranded DNA duplex.
77
In addition to explaining the mechanism for cleaving the primary
substrates of Artemis:DNA-PKcs, this model also explains how less commonly
encountered double- to single-stranded boundaries are cut (Figure 5.2). Among
these, only the single-stranded DNA substrate must anneal to itself to form a
transient double-stranded substrate, and the short-lived nature of this annealing
explains why single-stranded DNA is cut much less efficiently by Artemis or
Artemis:DNA-PKcs (Gu et al., 2010). Various other substrates can also be
explained by our model, and, are cleaved at the predicted locations (Ma et al.,
2005a). The pseudo-Y, flap and bubble structures may all be encountered during
different stages of DNA replication or repair. Importantly, a free DNA terminus
(blunt-end, overhang, or hairpin) is required to activate the serine/threonine
kinase activity of DNA-PKcs (Meek et al., 2008). Therefore, if a flap or a bubble
structure is present internally and far removed from a DNA terminus, DNA-PKcs
will not be activated, and thus neither Artemis nor Artemis:DNA-PKcs will cut (Ma
et al., 2005a).

78
Figure 5.2. Artemis Activity on Other DNA substrates. We propose that Artemis maintains the
three recognition points on other substrates (ssDNA, pseudo-Y, 5’ flaps and symmetrical bubble
structures) by remodeling the end into “hairpin-like” structures. While less apparent, ssDNA has
the ability to fold back and base pair with itself to create the “hairpin-like” structures. Recognition
site A (blue dot) is located on the 5’ -> 3’ (top) strand at the ss/dsDNA boundary. Recognition site
B (green dot) is located on the 3’ -> 5’ (bottom) strand directly across from recognition site A. The
proposed action site (red arrowhead) is located 1-nt on the 5’ side of recognition site B as shown
in the figure.
CONTRIBUTION OF DNA-PKCS TO SUBSTRATE RECOGNITION
As discussed below, Artemis and DNA-PKcs form a tight complex. DNA-
PKcs is only active as a protein kinase when DNA termini, ATP and Mg
2+
are
present to permit autophosphorylation of DNA-PKcs, which then can stimulate
Artemis nuclease activity (Goodarzi et al., 2006; Gu et al., 2010; Ma et al., 2002).
However, using purified proteins, we and others have shown in vitro that the
divalent cation, Mn
2+
, enables Artemis to function independent of DNA-PKcs (Gu
et al., 2010; Huang et al., 2009). All of the substrate structure recognition that we
have described above applies not only to Artemis with DNA-PKcs, but also to
Artemis activity in the presence of Mn
2+
without DNA-PKcs. This means that the
substrate distortion into a hairpin-like configuration is likely due to Artemis alone.
It is thought that the C-terminus of Artemis acts as a regulatory region since C-
terminal truncation mutants allow Artemis to be constitutively active independent
of DNA-PKcs (Niewolik et al., 2006). Thus, Mn2+ most likely interacts with
Artemis to permit Artemis to be constitutively active. In addition, the three key
recognition points of Artemis with the DNA substrate are independent of DNA-
PKcs.
What then is the role of DNA-PKcs in the Artemis:DNA-PKcs complex?
Under physiological conditions (with Mg
2+
but not Mn
2+
), Artemis is inactive
without an autophosphorylated DNA-PKcs (Goodarzi et al., 2006). Therefore,
79
DNA-PKcs is essential for Artemis activity in the presence of Mg
2+
. Somehow,
the Mn
2+
must change the conformation of Artemis so as to mimic the effect of an
autophosphorylated DNA-PKcs. DNA-PKcs requires a broken DNA end for it to
bind (usually with Ku) (Dvir et al., 1992; Jovanovic and Dynan, 2006), and this is
necessary for Artemis to be active in cutting the phosphodiester backbone at that
DNA end. Therefore, the reliance of Artemis on an activated DNA-PKcs makes
the Artemis:DNA-PKcs complex responsive to broken DNA ends.

ROLE OF ARTEMIS:DNA-PKCS IN V(D)J RECOMBINATION
V(D)J recombination relies on the RAG complex to bind and cut at
recombination signal sequences (RSS) adjacent to V, D, and J segments,
resulting in coding ends that are hairpinned and signal ends that are blunt (Figure
5.3). The hairpin at the coding end must be opened to be joined to form a coding
joint. Based on genetic and biochemical evidence, the hairpin opening at coding
ends of the V, D, and J segments during V(D)J recombination is completely
dependent on the Artemis:DNA-PKcs complex (Figure 5.3) (Li et al., 2002; Ma et
al., 2002). Since the Artemis:DNA-PKcs complex is located at each coding end at
the time of hairpin opening, the coding end resection is almost certainly due to
Artemis:DNA-PKcs. Indeed, the effect of coding end sequence on the extent of
resection in vivo matches quite well with the known DNA sequence effects by
purified Artemis:DNA-PKcs in biochemical studies (Gauss and Lieber, 1996; Ma
et al., 2004). In addition, the signal ends, which are the DNA fragments cut out
and circularized during deletional V(D)J recombination, occasionally suffer a
80
small amount of end resection, which is also due to Artemis:DNA-PKcs activity
(Touvrey et al., 2008). Thus, the evidence is very strong that the Artemis:DNA-
PKcs complex is the nuclease involved in all of the resection that occurs during
V(D)J recombination.

Figure 5.3. Artemis Opens the Hairpins Generated in V(D)J Recombination. V(D)J
recombination occurs at sequences called 12-RSS and 23-RSS (triangles in the figure), where
RSS designates recombination signal sequence. An RSS contains conserved heptamer and
nonamer sequence elements, separated by either 12 or 23 non-conserved base pairs, and hence
the designation 12-RSS and 23-RSS. One recombination event requires one 12-RSS and one
23-RSS, and this is called the 12/23 rule. In early lymphoid cells, the RAG complex (RAG1 and 2
along with the constitutively expressed HMGB1 protein) nicks and then hairpins the coding ends
at the V and J segments in the figure. Ku can bind to any of the four DNA ends. The
Artemis:DNA-PKcs complex then binds to the V and J hairpin ends and nicks the hairpins in a
manner that usually results in a 3' overhang. The ends can be processed further by Artemis:DNA-
PKcs complex and a polymerase to introduce diversity. The NHEJ ligase complex then ligates the
ends together. Some antigen receptor loci have not only V and J segments, but also D segments;
hence, the name V(D)J recombination.

Importantly, Artemis null mice and DNA-PKcs mutant mice have similar
phenotypes that are consistent with our biochemical model (Rooney et al., 2002;
Xiao et al., 2009). Cells and animals from both mutants show failure of hairpin
81
opening and failure of coding joint formation, despite normal RAG cutting and
completion of signal joint formation. Both also show sensitivity to ionizing
radiation. Notably, human Artemis mutant patients and the one known human
DNA-PKcs mutant patient show similar phenotypes to these mice (van der Burg
et al., 2009; Li et al., 2002; Rooney et al., 2002; Xiao et al., 2009). We note that
some level of leakiness (a low level of successful coding joint formation) was
observed in some strains of mice, depending on the amount of 129/SvJ strain
background (Rooney et al., 2002). The amount of leakiness drops to nearly zero
in strains with a pure C57BL6 background (Xiao et al., 2009). The C57BL6
Artemis mutant response to bone marrow transplants is more comparable to
children with Artemis SCID than the “leaky” 129/SvJ strain (Xiao et al., 2009).
Even in leaky strains of mice, the vast majority of coding joint formation is
blocked. In human Artemis null patients, the level of leakiness is nearly zero (Li
et al., 2002) [i.e., only a few B cell clones survive (reflecting exceedingly rare
coding joint formation) out of hundreds of billions of B cells that failed to survive
(reflecting a >99.99% failure of coding joint formation)].

ROLES OF ARTEMIS:DNA-PKCS IN NHEJ
NHEJ is required for the joining phase in V(D)J recombination, and NHEJ
relies heavily on Artemis:DNA-PKcs. Based on these two points, we have
assumed that Artemis:DNA-PKcs is the major nuclease for all NHEJ processes.
We have summarized elsewhere a list of other possible nucleases that might
function in NHEJ (e.g., MRN, CtIP, ExoI, WRN and FEN-1) (Pannunzio et al.,
82
2014). In NHEJ, Ku is the protein that recognizes DSBs and recruits the other
NHEJ factors (Figure 5.4) (Dvir et al., 1992; Lieber, 2010). It is one of the most
abundant non-histone proteins in mammalian cells (~400,000 molecules per cell)
and tightly binds to DNA ends (K
D
= 6 x 10
-10
M) (Griffith et al., 1992; Mimori and
Hardin, 1986). DNA-PKcs is also abundant (50,000 to 100,000 molecules per cell
in humans (Anderson and Carter, 1996; Anderson and Lees-Miller, 1992)), and
binds DNA termini well on its own (K
D
= 3 x 10
-9
M) but 100-fold tighter when Ku
is present (K
D
= 3.5 x 10
-11
M) (West et al., 1998). Artemis and DNA-PKcs form a
tight complex that is stable in vitro even at 1 M monovalent salt (Ma et al., 2002).
We have recently used quantitative Western blots to determine that the number
of Artemis molecules per human cell (Reh pre-B cells) is ~70,000. Therefore, the
ratio of Artemis to DNA-PKcs to Ku is approximately 1:1:4. The relative
abundance and tight binding of Artemis:DNA-PKcs makes it likely that it is the
primary nuclease for mammalian NHEJ and explains why activity is increased
when Ku is present (Chang et al., 2015).
In vivo experiments have shown that approximately 20% of DSBs caused
by ionizing radiation require Artemis for repair (Riballo et al., 2004). It is important
to note that NHEJ is an iterative process (Lieber et al., 2008; Ma et al., 2004,
2005b). When Artemis is present, it may participate in nearly all NHEJ joining
event. But when it is absent as in the SCID T
-
B
-
NK
+
patients (Li et al., 2002), only
a small subset of ionizing radiation-induced DNA ends may require Artemis, and
thus be unresolvable in its absence. Therefore, the 20% failure to join should be
considered a conservative estimate of joining event that would require Artemis in
83
wild type cells. In vivo studies have shown that Artemis is involved in the repair of
a subset of DSBs generated by various forms of ionizing radiation (X-ray, γ-ray,
and α-particles) and reactive oxygen species (ROS) (Riballo et al., 2004;
Woodbine et al., 2011). These DSBs result in heterogeneous end structures,
which make it difficult to determine the specific subset of ends that Artemis is
processing. More specific DSB-inducing agents such as neocarzinostatin (NCS)
and bleomycin have been shown to generate DSBs that are repaired by Artemis
(Goodarzi et al., 2008; Mohapatra et al., 2011). NCS generates DSBs with a 5’-
phosphate and 3’-phosphate or 3’-phosphoglycolate termini with 1- to 2-nt 3’
overhangs. Bleomycin generates a mixture of blunt-ended or 1-nt 5’-overhang
substrates with 5’-phosphates and 3’-phosphglycolate termini (Mohapatra et al.,
2011). It will be interesting to utilize other forms of DSB generating agents to
determine the specific subset of DNA ends that require Artemis for repair.
84

Figure 5.4. Artemis is Involved in DNA End Repair via the NHEJ Pathway. Natural causes of
pathologic double-strand breaks are expected to generate heterogeneous, incompatible DNA
ends with little or no terminal microhomology. Ku (red circle and red rectangle) is the most
abundant DNA end binding protein in eukaryotic cells and can slide onto DNA ends that have
diverse configurations (38). Once Ku is bound to the DNA end, it can improve the binding
equilibrium of the nuclease, polymerases and ligase of NHEJ. The nuclease, polymerases, and
ligase appear capable of binding to and functioning at a DNA end without Ku, but the binding to
the DNA end is tighter when Ku is present. The most clearly identified nuclease thus far is the
Artemis:DNA-PKcs complex. The polymerases for NHEJ include the POL X polymerases, pol µ
and pol λ (Povirk, 2006). The ligase complex of NHEJ consists of XLF, PAXX, XRCC4, and DNA
ligase IV (Ahnesorg et al., 2006; Buck et al., 2006; Ochi et al., 2015). The bottom portion of the
diagram shows four equally plausible outcomes for the joining (the junction is highlighted in a red
box). There are hundreds of other possible joining outcomes even for one pair of starting DNA
ends with the same end configuration as that shown. This heterogeneity in outcome is in addition
to the heterogeneity in the end configuration generated by the original breakage process at that
very same set of phosphodiester bonds within the DNA duplex. Thus, there is heterogeneity in
the generation of the broken DNA ends and heterogeneity in how these ends are repaired.

85
FUTURE DIRECTIONS
We now know the essential structural features for DNA substrates of
Artemis. These substrates have a stable or transient double- to single-strand
boundary. We have determined how the structural features determine where
Artemis will hydrolyze the phosphodiester backbone of each substrate. This
model also explains why some DNA substrates are cleaved more efficiently than
others. This knowledge will aid in the co-crystallization of Artemis with DNA and
with the understanding of Artemis action on its substrates, once a crystal
structure is determined (Ochi et al., 2014).

86
CHAPTER 6.
DIFFERENT DNA END CONFIGURATIONS
DICTATE WHICH NHEJ COMPONENTS ARE MOST
IMPORTANT FOR JOINING EFFICIENCY

ABSTRACT
The NHEJ pathway is a key mechanism for repairing dsDNA breaks that occur
often in eukaryotic cells. In the simplest model, these breaks are first recognized
by Ku, which then interacts with other NHEJ proteins to improve their affinity at
DNA ends. DNA-PKCS binds to the Ku:DNA end complex along with the
nuclease, Artemis, to promote resection of the ends. DNA polymerase µ and λ
have the ability to add nucleotides for DNA ligase IV to ligate the ends with the
additional factors, XRCC4, XLF, and PAXX. In vivo studies have demonstrated
the degrees of importance of these NHEJ proteins in the repair of dsDNA breaks,
but interpretations can be confounded by other cellular processes and by the
difficulty in creating the various types of DNA end configurations. In vitro studies
with NHEJ proteins have been performed to evaluate the resection,
polymerization, and ligation steps but a complete system has been elusive. We
have now developed an NHEJ reconstitution system that includes the nuclease,
polymerase, and ligase components to evaluate relative NHEJ efficiency and
analyze ligated junctional sequences for various types of DNA ends. We find that
87
different dsDNA end structures have differential dependence on the nuclease,
polymerase, and ligase components. The dependence of some end joining on
only Ku and XRCC4:DNA ligase IV allows us to formulate a physical model that
incorporates nuclease and polymerase components as needed.

INTRODUCTION
The mammalian genome is a vast target for genotoxic agents. It is
estimated that the genome undergoes approximately 100,000 alterations per
day, which can result in an estimated ten double-stranded breaks (DSBs) per day
(Jackson and Bartek, 2010; Lieber, 2010; Lieber and Karanjawala, 2004; Martin
et al., 1985; Rich et al., 2000). The NHEJ DNA repair pathway is required to
repair many of these DSBs.
The NHEJ pathway first begins with the toroid-shaped Ku heterodimer
(Ku70 and Ku80) binding to the free dsDNA ends. Ku then recruits other NHEJ
factors as needed (Downs and Jackson, 2004). Many components improve
NHEJ efficiency in vivo, but the only essential component for NHEJ is the DNA
ligase complex consisting of XRCC4:DNA Ligase IV (X4/LIV) in a 2:1 ratio that
facilitates the ligation of a 3ʹ-hydroxyl to a 5ʹ-phosphate at the partner DNA end
(Grawunder et al., 1998a, 1998b; Sibanda et al., 2001). More recently, NHEJ
accessory factors, XLF and PAXX have been discovered, which bind to DNA
Ligase IV and Ku, respectively (Ahnesorg et al., 2006; Buck et al., 2006; Ochi et
al., 2015; Roy et al., 2015; Xing et al., 2015). Both XLF and PAXX have been
shown to promote the ligation of DNA ends that do not have any terminal base
88
pairing or microhomology (Incompatible ends) (Ahnesorg et al., 2006; Ochi et al.,
2015; Roy et al., 2015; Xing et al., 2015). However, if the DNA ends require
processing (e.g., if they are incompatible), a nuclease or polymerase may
become essential. Artemis appears to be the major NHEJ nuclease (Chang et
al., 2015), though other nucleases, such as APLF (also called PALF) may
participate in limited cases, especially when Artemis is not present (Li et al.,
2011). Artemis has 5ʹ exonuclease activity by itself and acquires endonuclease
activity when complexed with autophosphorylated DNA-PKcs (Chang et al.,
2015; Li et al., 2014).
An NHEJ reconstitution system in which we can observe the joined
products directly following polyacrylamide gel electrophoresis (PAGE) would
provide information on the ligation of each strand of the duplex, allow us to
determine NHEJ efficiencies, and provide detailed mechanistic insight after
sequencing the junctions (Figure 6.1). One earlier direct gel NHEJ reconstitution
did not include a nuclease, but rather focused on the ability of the ligase complex
to ligate across gaps in either strand and the ability of polymerase mu and
lambda to add random nts, thereby providing new MH between two DNA ends
(Gu et al., 2007a). Another early reconstitution included all known nuclease,
polymerase and ligase components, but was not sufficiently efficient to permit
direct gel assessment of joining products and tested only one pair of DNA end
configurations (Ma et al., 2004). None of the reconstitutions have demonstrated a
role for DNA-PKcs. The study here is the first to direct gel NHEJ reconstitution
system that includes all major nuclease, polymerase and ligase components.
89

FIGURE 6.1. NHEJ reconstitution workflow. NHEJ proteins are added to labeled DNA
substrate and NHEJ products are resolved in a denaturing PAGE. NHEJ products are cut out and
PCR amplified. PCR products are TA cloned into pGEM-T vector and transformed into E. coli.
Vector is then isolated from colonies and the junctions are sequenced.

RESULTS
Resection-dependent Compatible DNA Ends Require Artemis and Are
Strongly Stimulated by Ku and DNA-PKcs
Ligation of DNA ends with MH of even 1 base pair can be more efficiently
ligated by the X4/LIV complex than blunt DNA ends. We wondered whether DNA
ends with 3ʹ overhangs without terminal, but with internal, MH would undergo
90
ligation. We incubated a 5’-radiolabled DNA substrate with a (CCC CTT TTT T -
3ʹ) overhang and a substrate with a (GGG G -3ʹ) overhang with Ku, DNA-PK
cs
,
Artemis, and X4/LIV, as indicated (Figure 6.2). These two DNA ends have the
potential of generating 1 to 4-bp MH. The 3ʹ end of the bottom strand of the
labeled duplex and the 5ʹ end of the bottom strand of the unlabeled duplex were
biotinylated so that we could block one end of each duplex by adding
streptavidin. The string of T’s in the 3ʹ overhang is intended to prevent base
pairing until the T’s are resected. In this paper, we use the phrase ‘resection-
dependent’ to refer to joining that depends on any loss of nts, regardless of DNA
strand polarity. Supporting this point, we observed that NHEJ ligation products
are not generated unless the reactions contain the Artemis nuclease (Figure 6.2,
lanes 2 – 4 and lane 6).
But pairwise or more complete reactions have measurable ligation.
Approximately 4% of the substrate is converted to ligated product when Artemis
and X4/LIV is present (Figure 6.2, lane 5). The addition of Ku does not change
NHEJ efficiency (Figure 6.2, lane 7). When Artemis and DNA-PKcs are incubated
with X4/LIV, NHEJ efficiency increases to 13% (Figure 6.2, lane 8). The addition
of Ku to DNA-PKcs, Artemis, and X4/LIV further increases NHEJ efficiency to
28% (Figure 6.2, lane 9). We describe this event as resection-dependent (RD)
compatible end NHEJ since ligation can occur efficiently, if the Artemis:DNA-
PKcs complex is present to efficiently remove the nonhomologous portion of the
overhang so that the region of MH can be used to stabilize the DNA ends for
ligation.
91

FIGURE 6.2. NHEJ of resection-dependent compatible 3ʹ overhang requires Artemis and is
strongly stimulated by Ku and DNA-PKcs. NHEJ proteins (50 nM Ku, 25 nM DNA-PKcs, 25
nM Artemis, 100 nM X4/LIV) were incubated for 60 minutes at 37 °C with 20 nM *HC101/102 and
20 nM pHC115/116 in a reaction buffer containing 200 nM streptavidin to bind to biotin (B) to
block one end of the DNA. In addition, (p) represents a 5ʹ phosphate, and the asterisk (*)
represents the radiolabel. NHEJ efficiencies are noted underneath.

X4/LIV Stimulates DNA-PKcs-independent Artemis Activity
We were curious why there was resection and NHEJ products in Artemis
and X4/LIV conditions without DNA-PKcs (Figure 6.2, lane 5). Artemis has
intrinsic 5ʹ exonuclease activity and endonuclease activity when complexed with
92
DNA-PKcs. We incubated the (CCC CTT TTT T -3ʹ) overhang substrate with
either Artemis alone; DNA-PKcs and Artemis; Artemis and X4/LIV; or DNA-PKcs,
Artemis, and X4/LIV (Figure 6.3A). It has been reported that the C-terminal
region of Artemis (aa485 – aa 495) may interact with the DNA binding domain of
DNA Ligase IV (De Ioannes et al., 2012; Malu et al., 2012; Ochi et al., 2015). We
find that Artemis resection of the 3’ overhang increases when X4/LIV is added
(Figure 6.3A, lane 2 vs. 4). We next tested whether the Artemis resection could
also be due to the presence of a second substrate that may be able to generate
a 3’ flap if the overhangs transiently form C-G base pairs prior to resection of the
dTs (Figure 6.3B). Indeed, Artemis resection of the 3ʹ overhang is stimulated by
the addition of X4/LIV, independent of a second partner substrate (Figure 6.3B,
lane 4).

FIGURE 6.3. Artemis resection of 3ʹ overhangs is stimulated by the X4/LIV. NHEJ proteins
were incubated for 60 minutes 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-PK
cs
and 50 nM Artemis (lane 3); 50 nM Artemis and 100 nM X4/LIV (lane
4); and 25 nM DNA-PK
cs
, 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-PK
cs
, 25 nM Artemis, and
100 nM X4/LIV as indicated
93
Resection-dependent Compatible Ends Rely on MH for NHEJ
We next wondered whether the NHEJ of RD-compatible ends utilized the
MH generated when the 3ʹ Ts in the overhang (CCC CTT TTT T -3ʹ) are resected
by the Artemis:DNA-PKcs complex. We incubated NHEJ proteins and the 3ʹ
overhang substrate (CCC CTT TTT T 3ʹ) with a partner 3ʹ overhang substrate
that has the potential to generate 1 to 4-bp MH in the overhangs (GGGG -3ʹ)
(Figure 6.4, lanes 2 – 6), 1 to 2-bp MH (GG -3ʹ) (Figure 6.4, lanes 7 – 11), 1-bp
MH (G -3ʹ) (Figure 6.4, lanes 12 – 16), or 0-bp MH (Figure 6.4, lanes 17 – 21).
NHEJ efficiency decreases as the MH decreased from 16 % with 4-bp MH, 6 %
with 2-bp MH, 1% with 1-nt MH, and <1 % with 0-bp MH (Figure 6.4, lane 6 vs.
11 vs. 16 vs. 21). These data suggest that NHEJ of RD-compatible ends utilizes
the Artemis:DNA-PKcs complex activity to resect the overhang to produce a
region of MH for ligation to occur.

94

FIGURE 6.4. NHEJ of resection-dependent compatible 3ʹ overhangs is strongly dependent
on microhomology. NHEJ proteins (50 nM Ku, 25 nM DNA-PKcs, 25 nM Artemis, 100 nM
X4/LIV) were incubated with 20 nM *HC101/102 and either 20 nM pHC115/116 (lanes 1 – 6),
pHC115/123 (lanes 7 – 11), pHC115/124 (lanes 12 – 16), or pHC115/120 (lanes 17 – 21) for 60
minutes at 37 °C in a reaction containing 200 nM streptavidin to bind to biotin (B) to block one
end of the DNA. In addition, (p) represents a 5ʹ phosphate, and the asterisk (*) represents the
radiolabel. NHEJ efficiencies are noted underneath.

Incompatible Ends Cannot be Joined in a Biochemical System Containing
Ku, DNA-PKcs, Artemis, and X4/LIV Complex Alone
We next attempted to increase the complexity of in vitro NHEJ by testing
substrates with no regions of MH in the overhangs, (i.e. incompatible ends).
Joining of a DNA end with a (CCC CTT TTT T -3ʹ) overhang to an end with a
95
(TTT T -3ʹ) overhang showed marginal levels of NHEJ (Figure 6.5A, lanes 7 – 9).
Similarly, NHEJ of a DNA end with a (CCC CTT TTT T -3ʹ) overhang with a blunt-
ended DNA end pair showed marginal levels of NHEJ (Figure 6.5B, lanes 7 – 9).
These data suggest that Artemis:DNA-PKcs resection activity alone is not
sufficient to promote NHEJ of fully incompatible DNA ends. In contrast, blunt-end
ligation required only Ku and X4/LIV (Figure 6.5C, lane 3). The addition of DNA-
PKcs actually inhibited ligation ~2-fold irrespective of whether Artemis is also
included (Figure 6.5C, lane 3 vs. 6 vs. 7 vs. 9). These data suggest that NHEJ of
blunt-ended substrates in our biochemical system proceeds by direct ligation
without any requirement for MH or terminal base pairing after resection.

FIGURE 6.5. The Ku, DNA-PKcs, Artemis, and X4/LIV complex is not sufficient for NHEJ of
incompatible DNA ends while blunt-ended DNA only requires Ku and X4/LIV. NHEJ proteins
(50 nM Ku, 25 nM DNA-PKcs, 25 nM Artemis, 100 nM X4/LIV) were incubated for 60 minutes at
37 °C in a reaction buffer containing 200 nM streptavidin to bind to biotin (B) to block one end of
the DNA. In addition, (p) represents a 5ʹ phosphate, and the asterisk (*) represents the radiolabel.
NHEJ efficiencies are noted underneath. A, DNA substrates used were 20 nM *HC101/102 and
20 nM pHC115/119. B, DNA substrates used were 20 nM *HC101/102 and 20 nM pHC115/120.
C, DNA substrates used were 20 nM *HC121/102 and 20 nM pHC115/120.

96
Pol µ but Not Pol λ Stimulates NHEJ of 3’ Incompatible DNA Ends
Since Ku, Artemis, DNA-PKcs, and X4/LIV were not sufficient for NHEJ of
incompatible 3ʹ overhangs, we tested whether Pol X family polymerases can
stimulate NHEJ. The Pol X family members consist of Pol µ, Pol λ, Pol β, and
terminal deoxynucleotidyl transferase (TdT). Pol β and TdT do not participate in
NHEJ, except for TdT’s role in NHEJ in pre-B or pre-T cells (Lieber, 2010). Pol µ
has the ability to add nucleotides in either a template-dependent or template-
independent manner (Moon et al., 2007). Conversely, Pol λ is primarily a
template-dependent polymerase, with much less template-independent activity
(Moon et al., 2007). The addition of Pol µ or Pol λ did not significantly increase
NHEJ of RD-compatible ends (Figure 6.6A, lanes 2 – 4 and Figure 6.6B, lanes 2
vs. 4). However, Pol µ was able to promote two ligation events, suggesting that
streptavidin does not completely block one end from ligation events and that Pol
µ may be promoting NHEJ (Figure 6.6A, lane 3 and Figure 6.6B, lane 4). These
data suggest that resection, and not nucleotide addition, is sufficient for NHEJ of
RD-compatible DNA ends but Pol µ may be able to add nts that would generate
occult MH (Lieber, 2010).
NHEJ of 3ʹ incompatible DNA ends increased from approximately 0.1 to
4.6% with the addition of Pol µ but not Pol λ (Figure 6.6A, lanes 6 – 8). A similar
increase from undetectable to 6.0% was observed with the addition of Pol µ in
the NHEJ of a 3ʹ overhang with blunt-ended DNA partner (Figure 6.6A, lanes 10
– 12). Furthermore, the role of Pol µ does not provide a DNA end stability role,
which is supported by the lack of NHEJ improvement upon Pol µ inclusion in
97
dNTP-free conditions (Figure 6.6B, lanes 2 – 4, 6 – 8, and 10 – 12). (Weaker
intensity bands that migrated between the substrate and NHEJ product are likely
DNA hairpinned products from NHEJ of two molecules of the identical duplex
followed by melting and intramolecular annealing of each strand (Gu et al.,
2007a). These data suggest that Pol µ may be adding nucleotides to help form
regions of MH for NHEJ of substrates with incompatible 3ʹ overhangs. Since Pol
λ does not stimulate NHEJ, the contribution of Pol µ most likely proceeds via its
template-independent addition.
A smaller effect was observed in the NHEJ of blunt-ended DNA. Pol µ only
marginally increased NHEJ from 14.2% to 20.4%, whereas, Pol λ did not change
NHEJ (Figure 6.6A, lanes 14 – 16). These data support our other findings
suggesting that blunt-ended NHEJ may proceed through direct ligation even
though the Artemis:DNA-PKcs complex, which is capable resecting nucleotides
from the blunt-ends, is present.

98

FIGURE 6.6. NHEJ of 3ʹ incompatible ends is stimulated by Pol µ but not Pol λ. NHEJ
proteins (50 nM Ku, 25 nM DNA-PKcs, 25 nM Artemis, 100 nM X4/LIV, 25 mM Pol µ, and Pol λ)
were incubated for 60 minutes at 37 °C in a reaction containing 200 nM streptavidin to bind to
biotin (B) to block one end of the DNA. In addition, (p) represents a 5ʹ phosphate, and the
asterisk (*) represents the radiolabel. NHEJ efficiencies are noted underneath. A, DNA substrates
used were 20 nM *HC101/102 and either 20 nM pHC115/116 (lanes 1 – 4), pHC115/119 (lanes 5
– 8), or pHC115/120 (lanes 9 – 12). Blunt-end ligations were performed with 20 nM *HC121/102
and 20 nM pHC115/120 (lanes 13 – 16). B, 100 uM dNTPs was incubated with DNA substrates
20 nM *HC101/102 and either 20 nM pHC115/116 (lanes 1 – 4), pHC115/119 (lanes 5 – 8), or
pHC115/120 (lanes 9 – 12).

PAXX, but not XLF, Stimulates NHEJ of blunt DNA ends
We next wondered whether XLF and PAXX, would improve joining in our
biochemical NHEJ system. We incubated XLF and/or PAXX with Ku, DNA-PKcs,
Artemis, X4/LIV, and Pol µ (Figure 6.7). We observed that XLF and PAXX have
no stimulation effect on the NHEJ of RD-compatible ends (Figure 6.7A, lanes 2 –
5). It is possible that at RD-compatible ends, other DNA ligase factors may be
unnecessary.
NHEJ of incompatible 3ʹ ends was not improved with XLF or PAXX (Figure
6.7B, lane 7 – 10). The same was observed for the NHEJ of DNA with a 3ʹ
overhang with a blunt-ended DNA partner (Figure 6.7B, lane 12 – 15). However,
99
NHEJ of blunt-ended DNA was stimulated ~2-fold by PAXX but not XLF (Figure
6.7B, lanes 12 – 15). We had already reported the finding that XLF does not
improve ligation (Gu et al., 2007b). These new data suggest that the role of the
PAXX may be to help align the 5ʹ-phosphate and 3’-hydroxyl by interacting with
Ku so that ligase can ligate efficiently.

FIGURE 6.7. PAXX only stimulates blunt-ended NHEJ. NHEJ proteins (50 nM Ku, 25 nM DNA-
PKcs, 25 nM Artemis, 100 nM X4/LIV, 500 nM PAXX, 200 nM XLF, and 25 mM Pol µ) were
incubated for 60 minutes at 37 °C in a reaction containing 200 nM streptavidin to bind to biotin (B)
to block one end of the DNA. In addition, (p) represents a 5ʹ phosphate, and the asterisk (*)
represents the radiolabel. NHEJ efficiencies are noted underneath. A, RD-compatible NHEJ was
performed with 20 nM *HC101/102 and 20 nM pHC115/116. B, DNA substrates used were 20 nM
*HC101/102 and either 20 nM pHC115/119 (lanes 1 – 5) and pHC115/120 (lanes 6 – 10). Blunt-
end ligations were performed with 20 nM *HC121/102 and 20 nM pHC115/120 (lanes 11 – 15).

100
NHEJ of a 5ʹ Overhang to a Blunt-end Is Stimulated by XLF and PAXX
Next we tested the effect of XLF and PAXX on the NHEJ of a 5ʹ overhang
substrate with the radiolabel on the blunt-ended 3ʹ end (Figure 6.8A). This
substrate can undergo head-to-tail, head-to-head, or tail-to-tail ligation events,
however both the head-to-head and tail-to-tail events will quickly form hairpins.
The hairpinned DNA will migrate much faster than the linear products formed
from head-to-tail events. Since we don’t observe any hairpinned bands between
the NHEJ product and substrate bands, we assumed that the preferred NHEJ
event was the (5ʹ TTT TTT CCC) head ligating to the blunt-ended tail end of the
substrate (Figure 6.8). XLF and PAXX marginally improved the low levels of
NHEJ observed in Ku and X4/LIV reactions (Figure 6.8B, lanes 2 – 5). In
reactions with Ku, Artemis, and X4/LIV, XLF and PAXX did not improve NHEJ
alone, but marginally improved NHEJ with XLF and PAXX together (Figure 6.8B,
lanes 6 – 9). However, NHEJ was stimulated individually by XLF and PAXX in
reactions containing Ku, DNA-PKcs, Artemis, and X4/LIV (Figure 6.8B, lanes 10
– 12). The addition of both XLF and PAXX together, increase NHEJ ~3-fold from
1% (XLF or PAXX alone) to 3% (XLF and PAXX) (Figure 6.8B, lanes 11 – 13).
These data suggest that XLF and PAXX are able to promote NHEJ of a 5’
overhang with a blunt-ended DNA partner. Since the ligation-product band
migrates approximately at the 152-nt position, it is likely that the 5ʹ overhang is
mostly processed prior to ligation (Figure 6.8B, lane 13). In addition, the NHEJ
products are larger when Artemis is not present (Figure 6.8B, lanes 3 – 5) or
when Artemis 5ʹ exonuclease activity is dominant in DNA-PKcs free conditions
101
(Figure 6.8B, lanes 6 – 9).
We next tested the role of Pol µ and Pol λ in the NHEJ of the 5ʹ overhang
to a blunt-end. Both Pol µ and Pol λ only marginally stimulated NHEJ (Figure
6.8C, lanes 2 – 4). These data suggest that the addition of nucleotides is not
used to join DNA with blunt-ends and 5ʹ overhangs.

FIGURE 6.8. NHEJ of 5ʹ overhang to a blunt-end is stimulated by XLF and PAXX with no
polymerase effect. NHEJ proteins (50 nM Ku, 25 nM DNA-PKcs, 25 nM Artemis, 100 nM
X4/LIV, 500 nM PAXX, 200 nM XLF, 25 mM Depiction of DNA substrate used (p) represents a 5ʹ
phosphate, and the asterisk (*) represents the radiolabel. Pol µ and Pol λ were incubated with 20
nM pJG277*/JG226-ddG for 60 minutes at 37°C. NHEJ efficiencies are noted underneath. A,
Depiction of DNA substrate used. (Head) refers to the end with the 5’ overhang and (Tail) refers
to the blunt-end. B, XLF and PAXX were varied in NHEJ reactions with Ku, X4/LIV (lanes 2 – 5),
Ku, Artemis, and X4/LIV (lanes 6 – 9), Ku, DNA-PKcs, Artemis, and X4/LIV (lanes 10 – 13). C,
Pol µ and Pol λ were varied in NHEJ reactions with Ku, DNA-PKcs, Artemis, X4/LIV, XLF, and
PAXX as noted.
102
NHEJ Sequences of RD-Compatible Ends Reveal a Preference for
Resection to Generate MH for Ligation
We next sequenced the junctions from the joining products by cutting out
bands from the PAGE gels, PCR amplifying, and TA-cloning them into a pGEM-T
vector for sequencing (Figure 6.1). We found that NHEJ of RD-compatible ends
are maximal in Ku, DNA-PKcs, Artemis conditions (Figure 6.2, lane 9; Figure
6.5A, lane 2; and Figure 6.6B, lane 2). The sequences from these junctions
showed that the preferred modification is to resect the 3’ dTs from the (CCC CCT
TTT TT -3’) overhang to create approximately 3- to 4-nts MH to stabilize ligation
(Table 6.1, RD-compatible ends and Table 6.2). Surprisingly, the addition of Pol
µ did not result in any sequences with additional nucleotides (Table 6.1, RD-
compatible ends).

NHEJ Junctions from the Joining of 3’ Incompatible Ends Reveals a
Preference for Resection and Nucleotide Addition to Provide MH for
Ligation
On the other hand, NHEJ of DNA ends with no potential microhomology
on the 3ʹ overhangs (incompatible 3’ ends) was very weak without Pol µ (Figure
6.6A, lane 7 and Figure 6.6B, lane 8). Hence, the preferred sequences were to
resect a varying amount of nucleotides from the (CCC CCT TTT TT -3ʹ) overhang
and to carry out synthesis of primarily dAs which was able to form 2- to 4-nts MH
with the 3ʹ (TTT T -3ʹ) on the other DNA end (Table 6.1, Incompatible 3ʹ ends
and Table 6.2). One unique sequence was the result of 3-bp MH and a ligation
103
over a G-T mismatch (Table 6.1, Incompatible 3ʹ ends). Not surprisingly, X4/LIV
was able to ligate over this mismatch (Gu et al., 2007a).

NHEJ Sequences of a 3’ Overhang with Blunt-ended Partner Reveals a
Preference for Resection and Direct Ligation Without MH
We next examined the junction sequences from the NHEJ of a 3ʹ
overhang ligated with a blunt-ended partner. NHEJ efficiency was best in
reactions that contained Pol µ (Figure 6.6A, lane 11 and Figure 6.6B, lane 12).
Sequences of these junctions revealed that the DNA ends undergo resection on
both ends of the DNA without significant nucleotide addition. Surprisingly, only
two out of the 13 sequenced junctions showed the addition of nucleotides (Table
6.1, 3ʹ overhang + blunt). Furthermore, only one of those sequences resulted
from a 3-bp MH with ligation over a G-T mismatch (Table 6.1, 3ʹ overhang +
blunt). Overall, the sequencing results indicate that the preferred repair process
is to resect the overhang and resect into the duplex without use of MH (Table
6.2).

NHEJ Sequences of Blunt-ended DNA Reveals Preference for Direct
Ligation
NHEJ of blunt-ended DNA is highly efficient process that only requires Ku
and X4/LIV for maximal activity (Figure 6.5C, lane 3). The addition of other NHEJ
factors does not improve NHEJ. Thus, the preferred joining product from two
blunt DNA ends is direct ligation (Table 6.1, Blunt + blunt and Table 6.2).
104
NHEJ Sequences of a 5’ Overhang with Blunt-ended DNA Reveals
Preference for Resection and Direct Ligation
NHEJ of a 5ʹ overhang to a blunt-end is maximal in Ku, DNA-PKcs,
Artemis, X4/LIV, XLF, and PAXX conditions (Figure 6.8B, lane 13). Ensuing
sequence analysis shows that the 5ʹ overhang is completely resected in most
joints without processing of the blunt-ended partner (Table 6.1, 5ʹ overhang +
blunt). Ligation then occurs without utilizing MH (Table 6.1, 5ʹ overhang + blunt
and Table 6.2).
105

TABLE 6.1. Sequence results of the most efficient NHEJ events. Sequencing results of the
NHEJ junctions with a column for proteins included, sequenced junctions, microhomology (MH)
utilized at the junction, and the number of molecules sequenced (n). Dashes (-) represent
resected bases, underscores (_) represent deletions, and bolded lowercase letters represent
added bases. Only the top strand is shown.

106

TABLE 6.2. NHEJ summary. The table describes the observed parameters for NHEJ of RD-
compatible ends, 3ʹ incompatible overhangs, 3ʹ overhang with a blunt-ended partner, blunt-ended
DNA, and 5ʹ overhang to a blunt-end.

DISCUSSION
Double-stranded DNA breaks arise from IR, ROS, replication errors,
inadvertent cleavage by nuclear enzymes, and by exogenous chemicals. These
breaks can create diverse DNA end structures that must be repaired. We have
developed an NHEJ reconstitution assay that can be used to compare NHEJ
efficiencies and analyze the junctions by analyzing sequences to determine the
effects of the proteins involved. It had been previously determined that the
ligation of compatible DNA ends is a highly efficient process that requires only
the X4/LIV complex and is stimulated by Ku when there is a 1-nt gap to ligate
over. Thus, we were now interested in determining how complex 5ʹ and 3ʹ
overhangs were processed and resolved. We find that NHEJ of incompatible
DNA ends is less efficient compared to NHEJ of compatible DNA ends. This
inefficient process is improved with the addition of Pol µ for 3ʹ incompatible ends.
PAXX alone promotes the NHEJ of blunt-ends, while PAXX and XLF
Artemis-dependent
NHEJ
Polymerase μ
s7mula7on
Polymerase λ
s7mula7on
XLF and PAXX
s7mula7on
Preferred modifica7ons
RD-compa7ble ends
++ - - -
• Resec(on to expose MH
3’ incompa7ble ends
++ ++ - -
• Resec(on
• Addi(on by Pol μ to create MH
3’ overhang + blunt
++ ++ - -
• Resec(on
• Addi(on by Pol μ
Blunt + blunt
- - -
+
(PAXX)
• Direct liga(on
5’ overhang + blunt
++ - -
+
(XLF and PAXX)
• Resec(on of 5’ overhang
• Liga(on without MH
107
synergistically enhance NHEJ of a 5ʹ overhang to a blunt-end. We have
summarized these observations in the diagram in Figure 6.9 (the red lines
represent known protein interactions and the red star represents interactions that
increase Artemis activity). The conclusions drawn from these experiments
support the flexibility and differing NHEJ protein requirements that we previously
hypothesized might exist (Pannunzio et al., 2014).

FIGURE 6.9. Diagram of end complex. This diagram shows how the various NHEJ proteins
associate at the ends. For simplification, we have only depicted the ligation of the top strand, but
the bottom strand will also undergo processing. Red stars represent interactions that stimulate
Artemis activity. Red lines represent known protein:protein binding interactions. Ku interacts with
LIV at the region containing the two BRCT domains (Costantini et al., 2007). The region between
the BRCT domains of LIV interacts with the helical domain of X4 (Grawunder et al., 1998a,
1998b; Sibanda et al., 2001). The N-terminal head domain of XLF interacts with the N-terminal
head domain of X4 (Ahnesorg et al., 2006). The C-terminus of PAXX interacts with Ku (Ochi et
al., 2015). The N-terminal BRCT domain of Pol µ interacts with the Ku:DNA complex (Ma et al.,
2004). The FAT domain in DNA-PKcs interacts with Ku (Spagnolo et al., 2006). Artemis is
activated by its interaction with DNA-PK
cs
through its C-terminus (aa 402 – 403). The C-terminus
of Artemis (aa 485 – 495) interacts with the N-terminal head domain of LIV (De Ioannes et al.,
2012; Malu et al., 2012; Ochi et al., 2013), and the current study indicates that this stimulates
Artemis activity (Figure 6.2).

108
Ku Contribution to NHEJ
We find that NHEJ of all DNA ends without MH require Ku. We previously
showed that NHEJ of ends with MH does not require Ku (Gu et al., 2007a), and
as the ends become more difficult to stabilize, perhaps by transient end
annealing, Ku is required. Ku has a high affinity for DNA ends (K
D
= 5.9 x 10
-10

M) (Mimori and Hardin, 1986) and can promote the binding of X4/LIV to the DNA
end when it is present (Nick McElhinny et al., 2000). This explains why Ku is
important for all of the substrates we test here in our study.

Artemis:DNA-PKcs Contribution to NHEJ
The Artemis:DNA-PKcs complex is required for hairpin opening of the
coding joints in V(D)J recombination (Ma et al., 2002). Consequently, both
Artemis and DNA-PKcs deficient cells fail to cleave this hairpin, consistent with
DNA-PKcs activation of Artemis (Lieber, 2010). In the context of NHEJ, Artemis-
deficient cells are radiosensitive but the majority of the DSBs in these cells are
repaired efficiently (Moshous et al., 2001; Nicolas et al., 1998). Approximately
20% of IR-induced DNA DSBs required Artemis for repair (Riballo et al., 2004).
Here, we find that Artemis activity is required for NHEJ of DNA substrates with
incompatible 5ʹ and 3ʹ overhangs. In contrast, blunt-ended DNA undergoes direct
ligation. The Artemis:DNA-PKcs complex is able to resect the overhang to reveal
regions of MH in RD-compatible ends (Table 6.1, RD-compatible ends). For DNA
ends with incompatible 3ʹ overhangs, resection occurs but that is not sufficient for
efficient NHEJ because there are no regions of MH (Figure 6.5A and B). Often,
109
the resection goes deeper into the duplex to allow exposure of other regions of
MH (Table 6.1). Artemis:DNA-PKcs activity on 5ʹ overhangs prefers resection of
the overhang to result in a blunt DNA end (Ma et al., 2002). Thus, the 5ʹ
overhang is processed to a blunt-ended substrate, in which direct ligation is
favored.
One surprising result was the observation of resection of the 3ʹ overhangs
in Artemis and X4/LIV conditions (Figure 6.2, lane 5; Figure 6.3B, lane 4; Figure
6.5A, lane 5; and Figure 6.5B, lane 5). It is possible that the reported interaction
of Artemis and DNA Ligase IV may also be able to activate Artemis activity (De
Ioannes et al., 2012; Malu et al., 2012; Ochi et al., 2013), though to a lesser
extent than by DNA-PKcs. DNA ligase IV may be able to stimulate Artemis at
DNA ends because X4/LIV has an affinity for ligatable DNA ends. (Hairpin DNA
ends likely do not bind X4/LIV.)
The dependence of some DSBs on DNA-PKcs and not other DSBs may
be relevant to immunoglobulin class switch recombination (Ig CSR). During Ig
CSR, DNA lesions generated by activation-induced deaminase (AID) lead to
DSBs. After the 5ʹ nuclease, ExoI, acts, we speculate that many 3ʹ overhangs
are common, though this may depend on which downstream Ig switch region is
being joined to the upstream Sµ DNA end. This would explain why DNA-PKcs is
variably required for some Ig switch recombinations, but not others (Bosma et al.,
2002; Franco et al., 2008).

110
Pol µ and λ Contribution to NHEJ
Pol X family members consist of Pol µ, λ, β and TdT. Pol β is involved in
base excision repair and lacks the BRCT domain that is responsible for its
interaction with Ku (Ma et al., 2004). TdT is only expressed in pre-B and T
lymphocytes and contributes to increasing immune diversity by adding random
nucleotides to coding ends in V(D)J recombination. Thus, TdT has no role in
NHEJ outside the context of V(D)J recombination. Pol µ is known to be involved
in NHEJ and has the ability to add nucleotides in a template-independent and
dependent manner (Gu et al., 2007a). Pol λ on the other hand, is primarily a
template-dependent polymerase. The structural differences in “loop 1” of these
polymerases, and in particular H329 within loop 1, have been implicated as the
flexible region that promotes template-independent activity (Moon et al., 2007).
Our study shows that Pol µ, but not Pol λ, plays a major role in promoting the
NHEJ of 3ʹ incompatible ends (Figure 6.6). Pol µ strongly stimulates NHEJ of 3ʹ
overhangs since nucleotides can be added that can form MH with the 3ʹ
overhang on the other DNA partner (Table 6.1). The lack of obvious regions of
MH in the NHEJ of 3ʹ incompatible overhangs reduces the ability of Pol λ to find a
stable template to initiate synthesis. In contrast, 5ʹ incompatible ends do not
require Pol µ (Figure 6.8C). The 5ʹ overhang is resected into a blunt-end
efficiently by the Artemis:DNA-PKcs complex. This blunt end then undergoes
direct ligation as is preferred for blunt-ends (Table 6.1).
In vivo studies of POL4 (homolog to mammalian Pol X family members)
mutants in yeast have also demonstrated that POL4 is indispensible for 3ʹ
111
overhangs but not for 5ʹ overhangs (Liang et al., 2016). Furthermore, Pol X family
members complement POL4 mutants, suggesting that the yeast model is
translatable to some aspects of mammalian cells (Daley and Wilson, 2005). Pol µ
has likely evolved to be able to add nucleotides in a template-independent
manner to help generate the MH required for ligation. Another study on C57BL/6
MEFs demonstrated that Pol µ was largely responsible for the nucleotide addition
in short (1- to 2-nt) 3ʹ incompatible ends (Pryor et al., 2015). The observation that
overhangs are resected by Artemis:DNA-PKcs followed by the addition of
nucleotides by Pol µ supports their importance in increasing junctional diversity
during V(D)J recombination.

XLF and PAXX Contribution to NHEJ
XLF and PAXX are the most recent NHEJ components discovered, and
these interact with XRCC4 and Ku, respectively (Ahnesorg et al., 2006; Dai et al.,
2003; Ochi et al., 2015). Single molecule studies have shown that
XRCC4/LIV/XLF may assemble at the DNA end with Ku (Reid et al., 2015). While
XLF deficient cells are more radiosensitive, biochemical evidence has shown that
XLF stimulates NHEJ of incompatible 3ʹ ends (Ahnesorg et al., 2006; Gu et al.,
2007b). XLF may only be required for the repair of a subset of DNA ends (Roy et
al., 2015). To date, PAXX has been shown to improve blunt-end ligation (Figure
6.7B) (Ochi et al., 2015). We find that XLF and PAXX stimulate NHEJ of a 5ʹ
overhang to a blunt-end (Figure 6.8B). XLF and PAXX do not seem to play a
112
significant role in the NHEJ of substrates with 3ʹ overhangs. The role of XLF and
PAXX may thus be to stabilize ends that do not utilize MH for ligation.

Concluding Comments
As summarized in Table 6.2, some DNA end configurations may be
ligatable with the involvement of a small number of NHEJ proteins, and others
require more NHEJ proteins. Our biochemical reconstitution supports this view,
which is consistent with in vivo studies.

113
CONCLUDING REMARKS
The DNA repair field has undergone a resurgence with the awarding of the
2015 Nobel Prize in Chemistry to Dr. Tomas Lindahl, Dr. Paul Modrich, and Dr.
Aziz Sancar for their contributions to the base excision, nucleotide excision, and
mismatch repair pathways, respectively. As I joined the Lieber lab, the
CRISPR/Cas9 genome-editing tool was first being characterized, and now
researchers are attempting to utilize it to edit the human genome. CRISPR/Cas9
technology utilizes the NHEJ pathway to repair the dsDNA breaks that are
formed during the editing process. The confidence in this new technology is in
part due to the fundamental knowledge of the NHEJ repair pathway.
Our studies into this repair pathway has led us to the development of a
reconstitution assay that can aid us in determining the efficiencies of NHEJ and
the preferred processes by analyzing the sequences. The previous system
developed in 2004 relied heavily on PCR and therefore, was not able to be used
to compare the efficiency of NHEJ events (Ma et al., 2004). In addition, we were
previously unable to find conditions in which DNA-PKcs was stimulatory in our
biochemical assays even though we know that it is important genetically. We find
that DNA-PKcs strongly activates Artemis activity, which in turn processes DNA
ends to increase the chance to form regions of MH for ligation to occur. We find
that Pol µ is the most important NHEJ protein for the resolution of incompatible 3’
overhangs. In addition, the ligase complex accessory factors, XLF and PAXX
seem to stimulate the NHEJ of incompatible 5’ ends, suggesting that their roles
114
are largely to stabilize the end complex since the action of Pol µ will primarily fill-
in the 5’ overhangs (Figure 6.9).
Our studies into the mechanism of NHEJ have generated a drug screen
effort targeting Artemis to treat blood cancers such as ALL. I had joined this
project at a crisis point, in which multiple outside labs had failed to scale Artemis
protein production to initiate the HTS. Ultimately, we had developed a scalable
protocol, which produced enough protein to complete the screen. The future of
this project is now to determine a set of lead candidates by more in vitro and in
vivo testing. In addition, it will be interesting to see if this project will yield a
crystal structure of the Artemis-inhibitor complex. The structural data will
hopefully be able to confirm our current model of Artemis recognizing three
structure-specific recognition points (Figure 5.1 – 5.2).

115
LIST OF ABBREVIATIONS
DSB – double-stranded break
NHEJ – Nonhomologous DNA end-joining
CSR – Class switch recombination
ALL – Acute lymphoblastic leukemia
HTS – High-throughput screen
IR – Ionizing radiation
ROS – Reactive oxygen species
Ig – Immunoglobulin
TCR – T cell receptor
AID – Activation-induced cytidine deaminase
RSS – Recombination signal sequence
RAG – Recombination activating gene protein
HMGB – High-mobility group box protein
HR – Homologous recombination
DNA-PKcs – DNA Protein Kinase catalytic subunit
MH – Microhomology
XRCC4 – X-ray repair cross-complementing protein 4
PIKK – Phosphatidylinositol 3-kinase-related kinase
ATM – Ataxia telangiectasia mutate
β-CASP – β-lactamase – CPSF, Artemis, SNM1, and PSO2
CPSF – Cleavage and polyadenylation specificity factor
SNM – Sensitive to nitrogen mustard
116
PSO – Psoralen
TdT – Terminal deoxynucleotidyl transferase
BRCT – BRCA1 C-Terminal
XLF – XRCC4-like factor
PAXX – Paralog of XRCC4 and XLF
MLPCN – Molecular Libraries Probe Production Centers Network
Sf9 – Spodoptera frugiperda
HEK – Human embryonic kidney
NTA – Nitrilotriacetic acid
DEAE – Diethylaminoethyl
CRO – Contract research organization
SEC – Size exclusion chromatography
FPLC – Fast protein liquid chromatography
WCL – Whole cell lysate
MRN – Mre11/Rad50/NBS1 complex
WRN – Werner syndrome, RecQ-like helicase
FEN-1 – Flap structure-specific endonuclease 1

117
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Abstract (if available)

Abstract Pathological DNA double-strand breaks (DSBs) are one of the most harmful forms of DNA damage, since both of the DNA strands are broken. These breaks can result in cell death from the loss of a chromosomal arm or by promoting the apoptotic pathway. In mammalian cells, DSBs are repaired predominantly by the nonhomologous DNA end-joining (NHEJ) pathway. Physiological DSBs are created during V(D)J recombination and class-switch recombination (CSR) and require the NHEJ pathway to resolve these breaks. Thus, defects in NHEJ result in marked sensitivity to ionizing radiation and the ablation of lymphocytes. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes, but which might also contribute to some of the genetic alterations that cause aging and cancer. This negative feature is offset by the flexibility of NHEJ proteins in handling many types of DNA end configurations and many types of damage. Mechanistic studies have revealed that the NHEJ pathway can be targeted for the treatment of cancers that utilize this pathway. Artemis is a structure-specific NHEJ nuclease that is required to open the DNA hairpin intermediates in V(D)J recombination. Thus, inhibiting Artemis can be used as a first-in-class nuclease inhibitor for the treatment of blood cancers that rely of V(D)J recombination, such as acute lymphoblastic leukemia (ALL). The development of a high-throughput screen (HTS) for Artemis inhibitors has required the scaling and optimization of Artemis purification. In addition, we use biochemical assays to unify Artemis activity to propose a function-based model along with the development of a direct gel NHEJ reconstitution assay to determine the contribution of NHEJ factors in DSB repair.

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Characterization of three novel variants of the MAVS adaptor

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Multiple functions of the PR-Set7 histone methyltransferase: from transcription to the cell cycle

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Targeting glioma cancer stem cells for the treatment of glioblastoma multiforme

Asset Metadata

Creator Chang, Howard Hoyon (author)

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

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 10/06/2016

Defense Date 09/14/2016

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

Tag Artemis,DNA repair,double-stand breaks,dsDNA,high-throughput screen,NHEJ,non-homologous DNA end-joining,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lieber, Michael R. (committee chair), Zandi, Ebrahim (committee chair), Hsieh, Chih-Lin (committee member)

Creator Email hochang1@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-312862

Unique identifier UC11213739

Identifier etd-ChangHowar-4867.pdf (filename),usctheses-c40-312862 (legacy record id)

Legacy Identifier etd-ChangHowar-4867.pdf

Dmrecord 312862

Document Type Dissertation

Format application/pdf (imt)

Rights Chang, Howard Hoyon

Type texts

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

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

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA