Download Double-Strand Break Repair

Molecular Life Sciences
DOI 10.1007/978-1-4614-6436-5_63-3
# Springer Science+Business Media New York 2014
Double-Strand Break Repair
Gilbert Chu*
Departments of Medicine and Biochemistry, Stanford University School of Medicine, Stanford, CA, USA
Synopsis
DNA double-strand breaks (DSBs) are the most dangerous form of DNA damage and can lead to
death, mutation, or malignant transformation. Mammalian cells use three major pathways to repair
DSBs: homologous recombination (HR), classical nonhomologous end joining (C-NHEJ), and
alternative end joining (A-NHEJ). Cells choose among the pathways by interactions of the pathways
with CtIP and 53BP1.
HR is restricted to S and G2 phases of the cell cycle, utilizing homologous newly replicated DNA
for error-free repair. Inherited mutations in the HR genes BRCA1, BRCA2, and PALB2 cause
susceptibility to breast, ovarian, and pancreatic cancer. C-NHEJ operates during all phases of the cell
cycle and during V(D)J recombination of the antibody and T-cell receptor genes, joining DNA ends
while minimizing nucleotide loss and addition for error-prone repair. Inherited mutations in the
C-NHEJ genes DNA-PKcs, Artemis, and XLF cause the syndrome of radiation sensitivity with
severe combined immunodeficiency. A-NHEJ is a backup pathway for C-NHEJ, joining DNA ends
after resection back to regions of microhomology for error-prone repair. A-NHEJ is responsible for
creating the chromosomal translocations seen in cancer. Thus, understanding DSB repair pathways
will lead to insights for immunology and cancer.
Introduction
DNA double-strand breaks (DSBs) pose grave problems for the mammalian cell. Unrepaired breaks
lead to death. Inaccurate repair generates mutations or chromosome translocations, which can lead to
cancer.
DSBs arise from many sources. Exogenous sources include ionizing radiation and topoisomerase
II inhibitors. Ionizing radiation produces ends with non-ligatable nucleotides containing ruptured
ribose rings or aberrant chemical groups, such as 50 -hydroxyl, 30 -phosphate, or 30 -phosphoglycolate
groups. The damaged nucleotide must be either repaired or removed prior to ligation. Topoisomerase II inhibitors include the anticancer drugs etoposide and doxorubicin. Topoisomerase II untangles
newly replicated DNA by cutting both strands of one DNA double helix to allow a second DNA
double helix to pass through. Topoisomerase II inhibitors trap the enzyme as a protein-bridged DSB
intermediate.
Endogenous sources of DSBs include V(D)J recombination and class switch recombination.
V(D)J recombination generates DSBs with blunt ends and unusual hairpin ends in order to create a
diverse repertoire of B-cell antibodies and T-cell receptors that recognize foreign antigens. The brief
definition entitled “▶ V(D)J Recombination” provides additional information. Class switch recombination switches the constant region of an antibody while preserving its variable region. After class
*Email: [email protected]
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switching, the antibody recognizes the same antigen but interacts with a different effector molecule
to activate a specific immunological response.
Befitting the danger of DSBs, eukaryotic cells have evolved three repair pathways (Fig. 1).
Homologous recombination (HR) occurs during S and G2 phases of the cell cycle, utilizing
homologous DNA (usually from the newly replicated sister chromosome) to repair DSBs. HR is a
conservative repair pathway because it restores the DNA sequence even if the source of the DSB
disrupts nucleotides near the ends. Classical nonhomologous end joining (C-NHEJ) joins compatible DNA ends precisely and noncompatible DNA ends with limited nucleotide deletion or addition.
C-NHEJ is nonconservative but optimizes the preservation of DNA sequence. Alternative
nonhomologous end joining (A-NHEJ) repairs DSBs by deleting nucleotides back to regions of
microhomology, creating junctions with deletions generally larger than those from C-NHEJ. Thus,
A-NHEJ is the least conservative pathway.
Prokaryotes and eukaryotes differ significantly in how they perform DSB repair. However, the
eukaryotic DSB repair pathways are conserved from yeast to mammals. This review emphasizes the
mammalian pathways and highlights their relevance for human disease.
Choice of the Pathway for Repairing DSBs
Choice of the repair pathway can depend on the source and timing of the DSB. V(D)J recombination
generates two DSBs and funnels their joining toward C-NHEJ (Dudley et al. 2005). Class switch
recombination generates two DSBs that are joined by both C-NHEJ and A-NHEJ (Yan et al. 2007).
In S and G2 phases of the cell cycle, cyclin-dependent kinase CDK2 directs DSBs toward repair
by HR.
MRN (Mre11-Rad50-Nbs1) accumulates rapidly at sites of DSBs, holding the two DNA ends
together (Fig. 2) (Stracker and Petrini 2011). Consistent with its role in tethering DNA ends, MRN
has an overall stoichiometry of Mre112-Rad502-Nbs12. The proteins in the MRN complex have
distinct biochemical activities. Rad50 contains a globular domain and an extended coiled-coil
ending in a hook domain that facilitates homodimerization of two Rad50 molecules. Since Rad50
interacts with Mre11 and Nbs1, Rad50 homodimerization generates the full MRN complex. Mre11
can tether two DNA ends to each other and has endonuclease and 30 –50 exonuclease activities
Fig. 1 Pathways for DSB repair. Homologous recombination (HR) utilizes homologous DNA (red) to conserve DNA
sequence. Classical nonhomologous end joining (C-NHEJ) minimizes loss of DNA in DNA sequence (black).
Alternative NHEJ (A-NHEJ) deletes nucleotides back to regions of microhomology, leaving one copy of the
microhomology region (black box)
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Fig. 2 Choice of pathway for DSB repair. MRN tethers the DNA ends to each other and activates the ATM kinase.
ATM phosphorylates its target proteins to coordinate the cellular response to the DSB. Binding of 53BP1 blocks end
resection and channels repair toward C-NHEJ, which is initiated by binding of Ku to the DNA ends. Binding of CtIP to
MRN leads to A-NHEJ or HR. In S and G2 phases of the cell cycle, cyclin-dependent kinase CDK2 phosphorylates CtIP,
committing repair to HR
in vitro. The exonuclease activity appears paradoxical, since the search for homologous sequence
during HR requires DNA with a 30 overhang. Indeed, other nucleases catalyze bulk 50 –30 resection,
as described below.
After binding to DNA ends at the DSB, MRN recruits ATM (ataxia telangiectasia mutated), a
serine-threonine protein kinase (Stracker and Petrini 2011). ATM kinase phosphorylates a number of
target proteins, leading to induction of cell cycle checkpoints, chromatin remodeling, and activation
of DNA repair.
Mutations in the genes encoding the MRN and ATM proteins cause a group of related human
diseases, consistent with their interaction with each other. ATM mutations cause ataxia telangiectasia (Savitsky et al. 1995), an autosomal recessive disorder characterized by poor coordination
(ataxia), dilation of small blood vessels (telangiectasia), cellular sensitivity to ionizing radiation,
immunodeficiency, and high risk for lymphoma and leukemia. Hypomorphic (partially inactivating)
recessive mutations in Mre11 cause ataxia telangiectasia-like disorder (ATLD) (Stewart et al. 1999),
manifesting ataxia and radiation sensitivity, but distinguished from ataxia telangiectasia by normal
immunity, normal cancer risk, slower disease progression, and absence of telangiectasias.
Hypomorphic Nbs1 mutations cause Nijmegen breakage syndrome (NBS) (Carney et al. 1998;
Varon et al. 1998), which is characterized by ▶ microcephaly, facial abnormalities, short stature,
▶ immunodeficiency, radiation sensitivity, and high risk for lymphoma.
CtIP (CtBP [carboxy-terminal-binding protein]-interacting protein) was originally identified as a
cofactor for the transcriptional repressor CtBP (You and Bailis 2010). Activation of the ATM kinase
permits CtIP recruitment to DSBs where it interacts with the Nbs1 component of MRN. The
interaction between CtIP and MRN facilitates repair by either A-NHEJ or HR (Fig. 2). CtIP
undergoes phosphorylation by CDK2 during S and G2 phases of the cell cycle, channeling the
DSB toward repair by HR.
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Table 1 Proteins involved in choosing the pathway for double-strand break repair
Protein Biochemical activity
Ku
DNA end binding
Mre11 Nuclease component of MRN complex (Mre11Rad50-Nbs1)
Rad50 Weak ATPase, component of MRN complex
Nbs1
ATM
CtIP
Human disease
Ataxia telangiectasia-like disorder (Stewart et al. 1999)
Nijmegen breakage syndrome-like disorder (Waltes
et al. 2009)
Nuclear localization, component of MRN complex Nijmegen breakage syndrome (Carney et al. 1998; Varon
et al. 1998)
Protein kinase, signaling of the presence of DSBs Ataxia telangiectasia (Savitsky et al. 1995)
DNA binding, promotion of MRN nuclease activity
Table 2 Proteins involved in homologous recombination
Protein
Mre11-Rad50Nbs1
RPA
Exo1
DNA2
BLM
BRCA1
BARD1
BRCA2
DSS1
PALB2
Rad51
Biochemical activity
See Table 1
Single-stranded DNA binding
Exonuclease
Helicase, endonuclease
Helicase, partner of EXO1 and DNA2
Ubiquitin ligase
Partner of BRCA1
DNA binding, loading of Rad51 onto ssDNA
Partner of BRCA2
Partner and nuclear localizer of BRCA2,
binding to BRCA1
DNA binding, ATPase, formation of
nucleoprotein filament
Human disease
See Table 1
Hereditary nonpolyposis colon cancer (Wu et al. 2001)
Bloom’s syndrome (Ellis et al. 1995)
Hereditary breast and ovarian cancer (Hall et al. 1990)
Hereditary breast and ovarian cancer (Wooster
et al. 1994)
Split-hand/split-foot syndrome (Crackower et al. 1996)
Familial breast or pancreatic cancers (Jones et al. 2009;
Rahman et al. 2007)
The p53-binding protein 1 (53BP1) also affects the choice of repair pathway (Panier and Boulton
2014). DSBs activate the ATM signaling cascade, which leads to the phosphorylation of histone 2A
variant H2AX together with the recruitment of 53BP1 to the damaged chromatin. Binding of 53BP1
to damaged chromatin blocks end resection at DSBs, preserving the DNA ends for binding by Ku
and thus channeling repair toward C-NHEJ. Table 1 summarizes the proteins involved in choosing
the DSB repair pathway.
Homologous Recombination (HR)
Table 2 summarizes the HR proteins. The MRN/CtIP complex resects DNA ends, including ends
with covalent modifications or bulky adducts (Buis et al. 2008; Sartori et al. 2007). MRN/CtIP
resects no more than 50–110 nucleotides in the 30 –50 direction, and this nuclease activity is
dispensable for unblocked DNA ends. However, ATM-mediated phosphorylation of CtIP together
with BRCA1 (breast cancer susceptibility type 1)-mediated ubiquitination of CtIP leads to the
extended 50 –30 resection of 100–200 nucleotides to form the 30 overhang required for homology
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searching during HR (San Filippo et al. 2008). Extended resection requires MRN, RPA (replication
protein A), the nucleases Exo1 and DNA2, and the helicase BLM (Bloom’s syndrome protein)
(Nimonkar et al. 2011).
Bloom’s syndrome is an autosomal disorder characterized by short stature, rash upon sun
exposure, and a high risk for cancer, including leukemia, lymphoma, and carcinoma. Bloom’s
syndrome cells show a strikingly elevated frequency of exchange events between homologous
chromosomes (sister chromatid exchanges).
Neither Exo1 nor DNA2 can generate the extensive 30 overhang required for HR when acting as a
purified protein in isolation. Exo1 has a relatively weak 50 –30 exonuclease activity. DNA2 has
endonuclease activity that will degrade either a 50 or 30 end. However, each nuclease forms a
complex with partner proteins to form an independent resection machine: BLM-DNA2-RPAMRN or Exo1-BLM-RPA-MRN (Nimonkar et al. 2011).
In the DNA2-dependent resection machine, DNA2 and BLM act together, utilizing the helicase
activity of BLM to enhance the endonuclease activity of DNA2 (Fig. 3). RPA, which binds to
ssDNA, enforces 50 polarity. In the Exo1-dependent resection machine, BLM and MRN increase the
affinity of Exo1 for DNA ends. MRN and RPA increase the processivity of Exo1 resection.
Fig. 3 Model for HR. During S and G2, CDK2 phosphorylates CtIP. BRCA1 then binds and ubiquitinates CtIP. The
DNA ends undergo extended 50 –30 resection catalyzed by BLM-DNA2-RPA-MRN or Exo1-BLM-RPAMRN. Resection generates 30 ssDNA tails of 100–200 nucleotides, which are protected by RPA binding. BRCA1
recruits PALB2 and BRCA2, which load Rad51 onto the ssDNA, displacing RPA to form a Rad51 nucleoprotein
filament. Rad51 mediates strand exchange, forming a D-loop (named for the shape of the DNA structure). Strand
exchange creates a primer for DNA synthesis on the homologous DNA template. The homology-guided reaction
restores the broken DNA to its original sequence
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In both resection machines, RPA prevents the ssDNA from folding over and hybridizing to itself
to form secondary structures. The intrinsic Mre11 nuclease activity of MRN is not required for either
machine. Rather, MRN provides a scaffolding function to recruit and stimulate the nuclease
activities of Exo1 and DNA2.
Both resection machines create DNA ends with 30 ssDNA overhangs of 100–200 nucleotides
coated with RPA. It is not clear why HR utilizes two different resection machines, particularly
because both machines include BLM, MRN, and RPA. However, the different activities of Exo1 and
DNA2 as isolated nucleases raise the possibility that they have distinct functions other than simple
end resection. For example, the endonuclease activity of DNA2 may allow removal of blocked
50 ends.
Following resection, several proteins cooperate to create a nucleoprotein filament that allows the
ssDNA to invade homologous DNA. BRCA1 and BRCA2 (breast cancer susceptibility type 2) play
key roles in loading Rad51 onto the ssDNA (Fig. 3). CDK2-mediated phosphorylation of Ser327 in
CtIP facilitates its binding to BRCA1, forming a ternary complex of CtIP, MRN, and BRCA1 (Chen
et al. 2008).
BRCA1 recruits BRCA2 to the DSB by binding to PALB2 (partner and nuclear localizer of
BRCA2), which in turn binds to BRCA2 (Sy et al. 2009; Zhang et al. 2009). Together with DSS1
(deleted in split-hand/split-foot syndrome), BRCA2 binds to DNA via two DNA-binding domains
(Yang et al. 2002). One domain binds to ssDNA and the other binds to double-stranded DNA,
positioning BRCA2 at the transition point from double-stranded DNA to the 30 ssDNA overhang.
Inherited mutations in BRCA1, BRCA2, and PALB2 confer susceptibility to breast, ovarian, and
prostate cancer (see the brief definition “▶ Hereditary Breast and Ovarian Cancer and Poly
(ADP-Ribose) Polymerase Inhibition”).
BRCA2 and PALB2 load Rad51 onto the ssDNA, displacing RPA (Fig. 3). Rad51 exists as a
heptamer in solution but upon binding to ssDNA forms a nucleoprotein filament with six Rad51
monomers per helical turn (San Filippo et al. 2008). Rad51 has an ATPase activity that permits
turnover via dissociation from the DNA.
Rad51 is the recombinase that mediates strand exchange for HR. The nucleoprotein filament of
Rad51-coated ssDNA captures a double-stranded DNA molecule and searches for homologous
sequence (Fig. 3). BRCA2 and PALB2 act synergistically to stimulate Rad51-mediated strand
exchange and D-loop formation (Buisson et al. 2010; Dray et al. 2010). The invading 30 end of
the ssDNA primes DNA synthesis from the homologous DNA molecule, replacing DNA sequence
lost from the broken DNA molecule.
The HR pathway can also lead to the crossing-over between homologous DNA chromosomes.
Thus, HR-directed repair of a DSB during mitosis can lead to a detrimental loss of heterozygosity. In
addition to its role in resection, BLM forms a complex with topoisomerase IIIa to suppress crossingover during homologous recombination (Wu and Hickson 2003). This role may explain why
Bloom’s syndrome cells have a high rate of sister chromatid exchange.
Classical Nonhomologous End Joining (C-NHEJ)
C-NHEJ repairs DSBs in all phases of the cell cycle by joining the broken ends directly without
using homologous DNA. DSBs may create ends with damaged nucleotides, which must be removed
before ligation can occur. Since C-NHEJ lacks a mechanism for replacing the damaged nucleotides,
the reaction is nonconservative. Table 3 summarizes the proteins involved in C-NHEJ.
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Table 3 Proteins involved in classical nonhomologous end joining
Protein
Biochemical activity
Ku
DNA end binding
DNA-PKcs DNA-dependent protein kinase,
synapsis of DNA ends
XRCC4
DNA binding, partner of ligase IV
Ligase IV
DNA ligase
XLF
DNA binding, promotion of
(Cernunnos) mismatched end ligation
PNKP
Polynucleotide kinase/phosphatase
Artemis
Endonuclease, exonuclease
Pol mu
Pol lambda
Human disease
Radiation sensitivity with severe combined immunodeficiency (van
der Burg et al. 2009)
Nijmegen breakage syndrome-like disorder (O’Driscoll et al. 2001)
Radiation sensitivity with severe combined immunodeficiency
(Ahnesorg et al. 2006; Buck et al. 2006)
Microcephaly and seizures (Shen et al. 2010)
Radiation sensitivity with severe combined immunodeficiency
(Moshous et al. 2001)
DNA polymerase
DNA polymerase
Initiation of C-NHEJ occurs when Ku (a heterodimer of Ku70 and Ku80) binds to the DNA ends
(Smider et al. 1994; Taccioli et al. 1994). Ku recruits DNA-PKcs (DNA-dependent protein kinase
catalytic subunit) to the DNA end, sliding to an inward position on the DNA (Yoo and Dynan 1999).
DNA-PKcs is an unusual serine-threonine protein kinase that undergoes activation on binding to
DNA (Jackson et al. 1990). Activation requires free DNA ends and may involve threading of
unpaired single strands into enclosed cavities in the DNA-PKcs structure (Hammarsten et al. 2000;
Leuther et al. 1999) and occurs upon formation of a synaptic complex containing two DNA ends and
two DNA-PKcs molecules (DeFazio et al. 2002). Subsequent DNA-PKcs autophosphorylation
generates conformational changes that dissociate DNA-PKcs from the DNA ends (Chan and
Lees-Miller 1996; Meek et al. 2008), which allows other C-NHEJ proteins to bind and process
the DNA ends.
Three proteins participate in the ligation reaction: XRCC4 (X-ray cross-complementing protein 4),
ligase IV, and XLF (XRCC-like factor). XRCC4 and ligase IV form a complex with DNA ligase
activity (Grawunder et al. 1997). XLF (also known as Cernunnos) interacts with XRCC4 (Ahnesorg
et al. 2006; Buck et al. 2006). XRCC4 and XLF are homodimers that interact with each other via their
head domains, and they are capable of forming a structure of alternating XRCC4 and XLF molecules
(Andres and Junop 2011; Hammel et al. 2011; Ropars et al. 2011; Wu et al. 2011). Ku, XRCC4/ligase
IV, and XLF have a mismatched end (MEnd) ligase activity that ligates any pair of DNA ends, even
ends with mismatched overhangs (Tsai et al. 2007). The proteins may form a megadalton protein-DNA
complex containing Ku plus a filament of alternating molecules of XRCC4/ligase IV and XLF (Tsai
and Chu 2013). The cooperative assembly of the filament may facilitate juxtaposition of the
mismatched ends for ligation.
MEnd ligase activity is particularly robust for 30 overhangs, which is relevant for V(D)J recombination, the pathway that generates immunological diversity (see the brief definition “▶ V(D)J
Recombination”). Recombination occurs by a pair of cleavages adjacent to two recombination
signal sequences. The cleavages are catalyzed by the endonuclease RAG1/RAG2 (recombinationactivating gene products 1 and 2) (McBlane et al. 1995). Each cleavage generates a blunt end
adjacent to signal sequence and a hairpin end adjacent to protein-coding sequence. C-NHEJ then
joins the blunt ends to each other in a precise reaction and the hairpin ends to each other in an errorprone reaction. In a complex with DNA-PKcs, the endonuclease Artemis opens the hairpin ends,
either with a nick at the tip of the hairpin or an asymmetric nick that creates a palindromic 30 singlestranded overhang (Ma et al. 2002). Terminal deoxynucleotidyl transferase may also add random
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nucleotides to the 30 end. The MEnd ligase complex can ligate a mismatched 30 overhang to preserve
the nucleotide sequences from the palindromic 30 overhang (P addition) or from the addition of
random nucleotides (N addition). Mutations in C-NHEJ genes cause several human diseases
(Table 3), including radiation sensitivity with severe combined immunodeficiency (RS-SCID),
where SCID is due to the importance of DNA-PKcs, Artemis, and XLF in V(D)J recombination.
In addition to ends ending in hairpins, C-NHEJ must join DNA ends with disrupted termini that
require enzymatic processing prior to ligation. Artemis has an intrinsic 50 –30 exonuclease activity.
Upon forming a complex with DNA-PKcs, Artemis acquires an endonuclease activity that cleaves 50
and 30 ssDNA overhangs (Ma et al. 2002). Polynucleotide kinase/phosphatase (PNKP) converts
50 -hydroxyl and 30 -phosphate DNA termini (such as those created by ionizing radiation) into
ligatable 50 -phosphate and 30 -hydroxyl ends. PNKP requires XRCC4, thus linking its processing
activity to ligation by ligase IV (Chappell et al. 2002; Koch et al. 2004; Mani et al. 2010). DNA
polymerases mu and lambda have been implicated in C-NHEJ by their interactions with Ku and
XRCC4. Both enzymes are recruited to fill gaps when ends have partially complementary overhangs
(Nick McElhinny et al. 2005).
C-NHEJ optimizes the preservation of DNA sequence by suppressing processing so that compatible DNA ends are joined precisely. For example, the blunt ends adjacent to V(D)J recombination
signal sequences are joined without nucleotide addition or deletion virtually 100 % of the time. In
human cell extracts that recapitulate C-NHEJ in intact cells, blunt and cohesive ends are joined
precisely, even though processing readily targets noncompatible ends to generate both nucleotide
addition and deletion (Budman and Chu 2005). These observations suggest that C-NHEJ controls
access of the processing enzymes to the DNA ends.
How does C-NHEJ prevent unnecessary processing of the ends? All polymerase and most
nuclease activity require the presence of XRCC4/ligase IV (Budman et al. 2007). The processing
enzymes polymerase mu, polymerase lambda, and PNKP interact with XRCC4, which appears to
grant access to the DNA ends. This mechanism ensures that XRCC4/ligase IV binds to the ends first
and catalyzes ligation if processing is not required. The small fraction of nuclease activity in the
absence of XRCC4/ligase IV could occur via recruitment and activation of Artemis by DNA-PKcs.
Thus, C-NHEJ regulates processing via an ordered series of steps: binding by Ku and then
DNA-PKcs, synapsis of the ends and DNA-PKcs autophosphorylation, release of the ends for
binding by XRCC4/ligase IV and XLF, and finally XRCC4-dependent recruitment of PNKP and
polymerase (Fig. 4).
Additional questions remain unanswered. Although Artemis participates in C-NHEJ, it is not
known if other nucleases also contribute to processing of the ends. The MRN complex tethers DNA
ends and contributes to end resection in A-NHEJ and HR. It is not known if MRN contributes to end
processing during C-NHEJ. Perhaps, the complex of MRN and CtIP resects DNA ends before Ku
binds to the ends, accounting for some of the deletions observed in C-NHEJ.
Alternative End Joining (A-NHEJ)
Evidence has accumulated for an alternative nonhomologous end-joining pathway (A-NHEJ)
(Deriano and Roth 2013). Deletion of XRCC4 or ligase IV reveals robust alternative end joining
during class switch recombination (Yan et al. 2007). Removal of portions of the RAG1 and RAG2
proteins reveals an alternative joining pathway during V(D)J recombination in NHEJ-deficient cells
(Corneo et al. 2007). In the absence of XRCC4/ligase IV, chromosomal translocations occur with
much greater frequency (Simsek and Jasin 2010).
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Fig. 4 Model for C-NHEJ. Ku binds to the DNA ends and slides inward upon recruiting DNA-PKcs to the end.
DNA-PKcs brings the ends together in a synaptic complex, which activates the DNA-PKcs kinase. DNA-PKcs
undergoes autophosphorylation and releases the ends for binding by XRCC4/ligase IV (XL). (The red dot indicates
where ligase IV interacts with XRCC4.) If the ends are ligatable with blunt or complementary overhangs, XL ligates the
ends directly. If 30 -phosphate or 50 -hydroxyl groups have damaged the ends, XRCC4 recruits PNKP to repair the
damage. XRCC4 and XLF assemble cooperatively into a protein-DNA filament to align the DNA ends, facilitating the
ligation of DNA ends even if they have noncomplementary overhangs. DNA polymerases mu and lambda and nuclease
activity process the ends
Table 4 Proteins involved in alternative nonhomologous end joining
Protein
Mre11-Rad50-Nbs1
CtIP
PARP-1
XRCC1
Ligase III
Biochemical activity
See Table 1
See Table 1
Poly(ADP-ribose) polymerase
DNA binding, partner of ligase III
DNA ligase
Human disease
See Table 1
A-NHEJ appears to be a distinct pathway, rather than a pathway cobbled together by substituting
another repair protein for a missing C-NHEJ protein (Deriano and Roth 2013). The junctions created
by A-NHEJ are not affected by whether Ku or XRCC4/ligase IV is absent. In the RAG mutants cited
above, A-NHEJ is robust even in the presence of intact C-NHEJ. Finally, A-NHEJ may have
evolved before C-NHEJ, since E. coli catalyze end joining by an A-NHEJ pathway but lack
C-NHEJ proteins, such as Ku. Table 4 summarizes the proteins believed to be involved in A-NHEJ.
The junctions created by A-NHEJ contain deletions, usually back to regions of microhomology.
These deletions are significantly longer than those seen in C-NHEJ. Thus, A-NHEJ is the least
conservative of the three repair pathways, serving as a backup system for HR and C-NHEJ.
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Fig. 5 Model for A-NHEJ. After limited resection by MRN/CtIP, the two ends are bound and held in a synaptic
complex by PARP-1. Nuclease activity removes the protruding 30 ends back to regions of microhomology (black boxes).
PARP-1 recruits XRCC1/ligase III to ligate the DNA ends
Apparently, DSBs pose such a threat that cells evolved A-NHEJ despite its error-prone nature. On
the other hand, it appears that A-NHEJ is primarily responsible for the chromosomal translocations
seen in cancer.
Some elements of the A-NHEJ pathway are now known (Fig. 5). Recruitment of CtIP to the MRN
complex activates Mre11 nuclease to resect the DNA ends (Lee-Theilen et al. 2011). Ligase III
promotes A-NHEJ during chromosomal translocation (Simsek et al. 2011). XRCC1 forms a tight
complex with ligase III (Caldecott et al. 1994), indicating that XRCC1 is also involved in A-NHEJ.
Poly(ADP-ribose) polymerase 1 (PARP-1) facilitates A-NHEJ during class switch recombination
(Robert et al. 2009). PARP-1, XRCC1, and DNA ligase III act together to join DNA ends. PARP-1
binds to ends, albeit with a lower affinity than Ku (Wang et al. 2006), consistent with a hierarchy that
favors C-NHEJ over A-NHEJ.
Features of A-NHEJ remain unclear. What regulates the choice between A-NHEJ and the other
pathways of HR and C-NHEJ? Upon binding to DNA, PARP-1 catalyzes transfer of 50–200
molecules of ADP-ribose from NAD+ to itself. Poly-ADP-ribosylation of PARP-1 results in its
dissociation from the DNA end. Does PARP-1 self-modification recruit other A-NHEJ proteins?
Conclusions
To mitigate the danger posed by DNA DSBs, cells have evolved three pathways for DSB repair. HR
is a conservative pathway that preserves DNA sequence, operating only in S and G2 phases of the
cell cycle when an undamaged sister chromosome is available for recombination. C-NHEJ joins
ends precisely if possible, but otherwise optimizes the preservation of DNA sequence. C-NHEJ
plays key roles in V(D)J recombination and class switch recombination. A-NHEJ joins broken ends
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not repaired by HR or C-NHEJ. Although this pathway may prevent death of the cell from an
unrepaired chromosome, it generates chromosome translocations that lead to cancer.
Acknowledgments
The author thanks Chun Tsai, Jian Fung, and Alex Chu for their helpful comments. This work was
supported by NIH grant 1R01GM086579.
Cross-References
▶ Classical and Alternative End Joining
▶ DNA Repair
▶ DNA Repair Polymerases
▶ Eukaryotic RecA Homologs
▶ Hereditary Breast and Ovarian Cancer and Poly (ADP-Ribose) Polymerase Inhibition
▶ Homologous Recombination in Lesion Bypass
▶ Ligases
▶ Mechanisms of DNA Recombination
▶ Mismatch Repair
▶ Nucleotide Excision Repair
▶ Recombination Regulatory Mechanisms
▶ Recombineering
▶ Regulation of DSB Repair by Cell-Cycle Signaling and the DNA Damage Response
▶ Single Strand Annealing
▶ Types of DNA Damage
▶ V(D)J Recombination
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