REVIEW

The control of DNA repair by the cell cycle

Nicole Hustedt and Daniel Durocher

The correct duplication and transmission of genetic material to daughter cells is the primary objective of the cell division cycle.
DNA replication and chromosome segregation present both challenges and opportunities for DNA repair pathways that safeguard
genetic information. As a consequence, there is a profound, two-way connection between DNA repair and cell cycle control. Here,
we review how DNA repair processes, and DNA double-strand break repair in particular, are regulated during the cell cycle to
optimize genomic integrity.

Maintenance of genome stability is essential for normal development,
cellular homeostasis and tumour suppression1. It relies on the precise
orchestration of DNA replication, chromosome segregation, DNA repair
and genome surveillance mechanisms, along with their integration with
cell cycle progression and myriad other processes such as transcrip-
tion and metabolism. The modulation of DNA repair by the cell cycle
is largely due to the fact that chromatin, the substrate of DNA repair,
undergoes major transitions as DNA is replicated, compacted and untan-
gled to allow for cell division. At the same time, DNA replication repre-
sents both a challenge and an opportunity for DNA repair.
The mitotic cell cycle is defined as the ordered series of events occur-
ring in distinct phases that ultimately results in the division of a cell into
two daughters2. Two key events punctuate the cell cycle: DNA replica-
tion, when the bulk of the nuclear genome is duplicated, defines S (syn-
thesis) phase; and mitosis, defined as the period in which chromosomes
are condensed, sorted and then equally distributed to daughter cells,
delineates M phase. Between the M and S phases are the gap phases G1
and G2, respectively 2. Cells can also enter quiescence (or G0), a state
of replicative dormancy. The metronome driving cell cycle progression
consists of the periodic activation and inactivation of cyclin-dependent
Ser/Thr kinases (CDKs)2. In mammals, the main cell cycle CDKs (CDK1,
CDK2, CDK4 and CDK6) partner with specific cyclins to form active
kinase holoenzymes. CDK1 pairs with cyclins A/B, CDK2 with cyclin
A/E and CDK4/6 interact with Dtype cyclins. CDK activity is generally
low in G1 and rises progressively until it reaches maximal activity upon
mitotic entry. Destruction of the A- and B-type cyclins mediated by
the anaphase-promoting complex cyclosome (APC/C), an E3 ubiquitin
ligase triggers mitotic exit, thereby re-establishing the G1 state.
The rise and fall of DNA double-strand break repair
The most dramatic example of DNA repair regulation by the cell cycle
is certainly the periodic inhibition of DNA double-strand break (DSB)
repair (Figs13). Two main groups of repair pathways mend DSBs: end
joining (EJ) and homologous recombination (HR). EJ pathways re-ligate
DNA ends together with some or no processing of the ends themselves.
They operate throughout interphase and are inhibited during the pro-
cess of mitosis. EJ pathways are subdivided into the non-homologous
end-joining pathway (NHEJ) (which is dependent on DNA ligaseIV
and Ku), and a group of less well elucidated alternative end joining
(A-EJ) pathways3,4 (dependent on DNA ligaseI/III) (Fig.1). While the
template-independent nature of NHEJ has led many to suggest that it
is error prone, it is probably accurate in the majority of cases5. The best
defined A-EJ pathway is theta-mediated end joining (TMEJ), where ends
processed to produce 3 overhangs are annealed at short stretches of
homologous sequences, as small as one nucleotide, to drive deletion of
the intervening sequences and ligation. TMEJ is dependent on PARP1,
DNA ligaseIII and the polymerase activity of DNA polymerase (POL,
encoded by POLQ)3,6. Evidence suggests that POL-mediated TMEJ acts
as a backup for both NHEJ and HR, since loss of POLQ is lethal in the
context of cells with mutations in both KU/53BP1 and BRCA1/BRCA2,
which impair NHEJ and HR, respectively 68.
In contrast, HR comprises repair systems that employ extensive
homology and templated DNA synthesis to regenerate the sequence
surrounding the break site9. The canonical HR pathway that mediates
the repair of two-ended DSBs is gene conversion (Fig.2), although HR
is also critical for the repair of one-ended DSBs, such as those produced
by broken replication forks9. As the ideal template for HR is the sister
chromatid, HR is restricted to the S and G2 phases of the cell cycle10.
HRmediated DSB repair requires the formation of extensive single-
stranded (ss) DNA through a process termed DNA end resection11 that
requires, for most types of ends, the action of CtIP (ref.12). The role of
CtIP is not yet completely understood, but based on studies in budding
yeast 13, it is believed to stimulate the endonuclease activity of MRE11
in complex with RAD50 and NBS1 (the MRN complex). After endo-
nucleolytic cleavage, resection proceeds bi-directionally 14,15, with the
exonuclease activity of MRN proceeding in the 35 direction, whereas

Nicole Hustedt and Daniel Durocher are at the Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5,
Canada. Daniel Durocher is also in the Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
e-mail: durocher@lunenfeld.ca

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Nuclear envelope to homology search, strand invasion and DNA polymerization, processes
by which the sequence surrounding the break site is resynthesized. This

CtIP
BRCA1 pathway generates joint DNA molecules, from D-loops to the four-way
Holliday junction (HJ). HR-mediated DSB repair is completed through
Exo1
RNF8 PTIP
the disassembly of these joint molecules. Extended D-loops can be
P BLM DNA2
RNF168 P
P directly unwound by helicases followed by ligation in a process termed

F1
MDC1 53BP1 synthesis-dependent strand annealing (SDSA)9. Alternatively, when

RI
P
P ATM
Me Me Ub
Ub both ends invade the template, double-HJs are formed. Double-HJs can
M N M N
undergo converging branch migration followed by the processing of the

P
CtIP
R R resulting hemicatenane. This process, termed HJ dissolution, is cata-
EXO1 lysed through the combined action of topoisomeraseIII and the RecQ
HELB RPA BLM DNA2
family helicase BLM bound to RMI1 and RMI2 (the BTR complex)19,20.
P ATM-dependent
Alternatively, nucleases can cleave HJs in a process termed resolution. In
P CDK-dependent
Limited end processing eukaryotes, the main HJ resolvases are the SLX4 complex (comprising
P PLK3-dependent
SLX4SLX1/MUS81EME1 and XPFERCC1) and the GEN1 nucle-
LIGI
LIGIII ase (Yen1 in budding yeast). BTR produces exclusively non-crossover
POLQ
formations that avoid inter-homologue exchanges and thus loss of het-
PARP1
Classical Alternative erozygosity 20,21. In the absence of BLM, cells show an increase in crosso-
non-homologous end joining vers, detectable by sister chromatid exchanges (SCEs) that depend on
end joining (TMEJ)
the structure-specific endonucleases MUS81EME1, SLX1SLX4 and
GEN122,23. When resection reveals direct repeats flanking the break site,
KU70 a RAD52-dependent process termed single-strand annealing (SSA)3,9
KU80
LIGIV can repair the break with the ensuing loss of the intervening sequences.
End joining products:
SSA is, by definition, error prone and thus resection must be balanced
525 bp homology
to promote gene conversion over SSA.

05 bp insertions/deletions Variable size insertion/deletions
NHEJ: ON
A-EJ: ON
HR: OFF (pre-RAD51);
ON (HJ dissolution)
OFF (HJ resolution)
G1
CDK activity
Figure 1 Repair of double-strand breaks in G1 and early Sphase. ATM
is recruited to DSBs through MRN and phosphorylates targets such as
nucleosomes (in particular H2AX, resulting in H2AX) (top left). H2AX
recruits MDC1, also an ATM target. MDC1 phosphorylation recruits the E3
ubiquitin ligase RNF8, which, through recruitment of a second E3 ubiquitin
ligase (RNF168), leads to histone H2A ubiquitylation. This modification,
together with H4K20 methylation, allows for 53BP1 recruitment (top
right). 53BP1 is phosphorylated at its Nterminus in an ATM-dependent
manner, creating docking sites for RIF1 and PTIP. 53BP1 blocks resection
at DSBs, thereby channelling DSB repair towards NHEJ. Long-range
resection is also limited through HELB. End joining can be divided into
two pathways: Ku- and DNA-ligase-IV-dependent classical non-homologous
end joining (NHEJ, left), and PARP1-, POL- and LigI/III-dependent DNA
polymerase-mediated end joining (TMEJ, right). The intensity of shading
in the grey band (bottom) indicates the magnitude of CDK activation in
G1/early Sphase. P, phosphorylation; Ub, ubiquitylation; Me, methylation;
red arrows, resection.
the redundant activities of EXO1 and the BLM/DNA2 nucleases cata-
lyse long-range 53 resection16. The ensuing ssDNA is first bound
by RPA, which must then be replaced by RAD51 to form a nucleofila-
ment competent for HR through the action of BRCA1, PALB2 and the
BRCA2 recombination mediator 17,18. The RAD51 recombinase is central
End resection gets a helping hand from CDKs
By virtue of being the first step shared by the HR and A-EJ processes, and
by being simultaneously inhibitory to NHEJ (in particular to Ku bind-
ing 24), DNA end resection is the critical node for the regulation of DSB
repair by the cell cycle25. The regulation of resection is often reformulated
in the context of DSB repair pathway choice, since resection helps set-
tle the competition between repair systems that has been observed in a
Early S wide variety of organisms2629. The importance of end stability in DSB
repair pathway selection was first recognized in budding yeast 30, and it
was there that CDK activity was found to be essential for activation of
DNA end resection31,32. The involvement of CDK in eukaryotic resection
is conserved in all species examined, including in human cells33.
The rising levels of CDK activity that characterizes Sphase entry
and progression results in the phosphorylation and activation of the
enzymes that catalyse end resection while simultaneously weakening the
impediments to this process. In budding yeast, Cdk1 (Cdc28)-dependent
phosphorylation of the CtIP homologue Sae2 on Ser267 (ref.34) stimu-
lates Mre11 endonuclease activity 13. Cdk1 also stimulates the ability of
the nucleosome remodeller Fun30 to antagonize the 53BP1 orthologue
Rad9 (ref.35), an inhibitor of end resection36. In vertebrates, CtIP is
phosphorylated on an equivalent residue, Thr847, which is critical for
the initiation of DNA end resection37. However, CtIP phosphorylation
is complex and several other residues are phosphorylated38,39, including
Ser327, which is targeted by CDKs to promote binding to BRCA140,41,
possibly to stimulate DNA end resection4244. Additionally, the CDK2-
dependent phosphorylation of Ser276 and Thr315 promotes binding to
the cis-trans prolyl isomerase PIN1 to dampen, rather than stimulate,
ssDNA formation45.
Whereas the phosphorylation of CtIP by CDKs is clearly involved
in the cell cycle regulation of end resection, as demonstrated by

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P highlight that additional levels of control must exist to enforce strict cell
HELB cycle regulation of resection and HR, and raise the question of how the
Nuclear enve
lope complex, multi-site phosphorylation of CtIP is integrated to produce the
optimal response to DSBs.
In addition to CtIP regulation, CDK activity promotes end resection
RNF8 PTIP
P P
through the phosphorylation of EXO148 and NBS14951. In budding yeast,
RNF168 P
Dna2 phosphorylation by Cdk1 is also involved in stimulating DNA end

F1
MDC1 53BP1

RI
P
ATM BRCA1
Me
Ub P Me Ub resection52, which suggests that each of the main complexes involved in

P
CtIP
M N the generation of ssDNA at DSB sites are positively regulated by CDK-
P

dependent phosphorylation.
R P
CDKs also promote DNA end resection by weakening the activity of
two distinct blocks to resection: one mediated by the chromatin-binding
P RPA
protein 53BP1 (explored in more detail below), and a second embod-
RPA
BLMDNA2 ied by HELB, an ssDNA translocase. HELB is recruited to DSBs via
EXO1
an interaction with RPA53,54, which suggests a feedback mechanism in
PALB2
BRCA1
which resected ends themselves prohibit resection. The Cterminus of
BRCA2 HELB contains dual nuclear import and export signals that are modu-
RAD51
lated by CDK2 phosphorylation such that nuclear HELB concentration
RAD51 decreases as CDK2 activity rises55,56. As the function of HELB requires
its nuclear localization53, these observations suggest that the HELB-
dependent block to resection weakens as Sphase progresses. Finally,
cyclins play direct, non-canonical roles in promoting HR: cyclinD1
TOPIIIA binds to RAD51 to promote its recruitment to DSBs57, while cyclinA2
BLM
HR products: R1 R2 upregulates MRE11 expression in Sphase by binding to its mRNA and
promoting its translation58.

Dissonance between Sphase and HR

Non-crossover Crossover Although it is often said that HR is activated in Sphase, this is not entirely
accurate: early studies clearly established that HR is suppressed in early
Sphase26,29,59,60. In one example26, NHEJ-defective Ku70 avian DT40
SLX4 EME1
cells are hypersensitive to ionizing radiation in G1 and early Sphase,
SLX1
MUS81 but become increasingly radioresistant upon replication of a large por-
GEN1 tion of their genome (mid-S onwards). Similar results were obtained by
enumerating H2AX foci in cells deficient in DNA-PKcs (ref.60) or by
NHEJ: ON
A-EJ: ON monitoring the recruitment of RAD5261. Together these experiments sug-
HR: ON (pre-RAD51); gest that HR activation and Sphase entry are uncoupled from each other.
ON (HJ dissolution)
This dissonance is not entirely surprising, because early S is similar to G1
OFF (HJ resolution)
in that only a small fraction of the genome is replicated. The question is
Late S G2
therefore whether there is a post-replicative and local activation of HR
CDK activity
behind the fork (Fig.4a), whether the local accumulation of pro-HR fac-
tors on replicated DNA induces global activation of recombination, or
Figure 2 Repair of double-strand breaks in late Sphase. Top: during Sphase,
CDK activity rises, resulting in phosphorylation of NBS1, HELB and CtIP. whether there is simply a timer that activates HR in mid-Sphase (Fig.4b).
NBS1 and CtIP phosphorylation stimulates MRN endonuclease activity and The simplest model for explaining HR activation during Sphase pro-
allows BRCA1 to bind to CtIP. BRCA1 counteracts 53BP1. Phosphorylation poses that replicated chromatin is competent for HR whereas unrepli-
of HELB results in its nuclear export. Middle: long-range resection proceeds
through the combined action of DNA2/BLM and EXO1, also a CDK target.
cated chromatin remains refractory to this type of repair (Fig.4a). The
BRCA1, BRCA2 and PALB2 promote replacement of RPA by RAD51 on recent discovery that the lack of methylation at the Lys20 residue of
ssDNA. Bottom: strand invasion, joint molecule and dHJ formation are steps histoneH4 (H4K20me0) represents a post-replicative chromatin mark62
of HR. dHJs can be dissolved by the BTR complex, resulting in non-crossover has a number of implications for this model. H4K20me0 is specifically
product formation; activity towards dHJ of the resolvases MUS81EME1 and
SLX1SLX4 separately is low. Another dHJ resolvase, GEN1, is excluded
recognized by the TONSL subunit of the MMS22LTONSL com-
from the nucleus. Red arrows, resection; green arrowheads, dHJ dissolution; plex 62, suggesting that this complex labels newly replicated chromatin.
brown arrowheads, dHJ resolution. MMS22LTONSL is a poorly characterized HR factor that promotes
RAD51 loading in response to DNA replication stress6365, suggesting
unscheduled activation of resection in G1 cells through the T847E phos- a direct link between post-replicative chromatin marking and HR. As
phomimetic CtIP mutant 37,46, there is also evidence that CtIP can be H4K20me0 is also incompatible with 53BP1 recruitment66,67, H4K20me0
active in G1 to promote A-EJ. Ser327 and Thr847 can be phosphorylated and its recognition may tip the balance of repair in favour of HR via two
by PLK3 in G1 cells independently of CDK activity 47. These observations independent means.

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In budding yeast, cohesin must be actively loaded at DSB sites to pro-

P
mote HR, as replication-coupled sister cohesion establishment is insuf-
RNF8 RNF168
ficient to promote repair 71,72. Cohesin factors are also loaded onto DSB
53BP1
sites in mammalian cells, suggesting that they also act at a late stage
P P P in DNA repair 73. Collectively, these observations suggest that cohesion
MDC1 improves the efficiency of the HR reaction rather than playing a direct
ATM
Me P Me role in its activation.
N M M N The post-replicative marking model of HR activation does not eas-
P

ily explain how DNA end resection is activated as Sphase progresses

CtIP

P R R
P

unless one invokes, for example, the titration of a resection inhibitor as

new chromatin is formed. One can rather envisage that the rise in CDK
activity seen in Sphase is interpreted as a sort of timer that triggers a
P P P rise in DNA end resection activity (Fig.4b). This behaviour could be
accomplished through the multisite phosphorylation of CtIP, coupled to
P

HELB No extensive the CDK2-dependent exclusion of HELB from the nucleus. The increase
PALB2 P
resection BRCA1 in HR factor abundance in Sphase may also contribute to an HR timer.
BRCA2
The resection timer concept is different from post-replicative marking
No RAD51 filament formation of chromatin because the former is probabilistic in nature whereas the
latter is deterministic with respect to DSB repair pathway choice.

TOPIIIA 53BP1 and BRCA1 battle for real estate

BLM
HR products:
R1 R2 53BP1 accumulates at DSB sites by recognizing nucleosomes modified
by methylation on histoneH4 Lys20 and ubiquitylation of H2A Lys15
by RNF16866,67,74. 53BP1 promotes NHEJ and opposes HR (and A-EJ)
at least in part by blocking DNA end resection. Limiting end resection
Non-crossover Crossover requires the ATM-dependent phosphorylation of the 53BP1 N-terminal
region, which in turn promotes the recruitment of PTIP and RIF17580,
which are both independently involved in blocking DNA end resection.
P P GEN1
The function of RIF1 in blocking end resection also involves MAD2L2
SLX4 EME1 (also known as REV7)81,82.
SLX1 MUS81 For DNA end resection to be activated in S/G2 cells, cells must weaken
the end-protection activity of 53BP1 in a manner that involves BRCA1
(Fig.2). Indeed, loss of 53BP1 in mice suppresses defective HR and the
NHEJ: OFF
lethality caused by loss-of-function mutations in Brca18385. As 53BP1
A-EJ: OFF (?) inactivation does not suppress the defects associated with mutations in
HR: OFF (pre-RAD51); other HR genes, these results imply that a major function for BRCA1
ON (HJ dissolution)
ON (HJ resolution) is to antagonize 53BP1. Further evidence for this model emerged when
Early mitosis Late mitosis
analyses of 53BP1 localization to DSB sites by structured illumination
CDK activity microscopy found that 53BP1 ionizing-radiation-induced foci were
reshaped by BRCA1 in S/G2 cells86. This may be due to the BRCA1-
Figure 3 Repair of double-strand breaks in mitosis. Top: RNF8 and 53BP1 mediated ubiquitylation of histoneH2A, which enables the nucleosome
phosphorylation in mitosis blocks their recruitment to DSBs. DSB ends may remodeller SMARCAD1 to antagonize 53BP187. BRCA1 also inhibits
be held together until their repair in the next G1 phase. Middle: resection is the recruitment of RIF1 to DSB sites77,78,88 in a manner that involves its
limited by HELB, and the phosphorylation of RPA and BRCA2 contributes
to inhibition of RAD51 replacement. Mitotic late-stage HR intermediates interaction with CtIP and the CDK-dependent phosphorylation of CtIP
must be removed before chromosome segregation. Bottom: CDK1-dependent at residue Ser32777. While some studies suggest that loss of RIF1 or REV7
phosphorylation promotes MUS81EME1SLX1SLX4 holocomplex suppresses the HR deficiency of BRCA1-mutated cells77,81, other studies
formation. GEN1 gains access to chromatin after nuclear envelope
instead point to PTIP being the key 53BP1 effector involved in opposing
breakdown. Unlike BTR-mediated dissolution, dHJ resolution results in
both crossover and non-crossover product formation, depending on the HJ BRCA1-dependent DSB repair 79.
cleavage orientation. Conversely, the recruitment of BRCA1 to DSB sites in G1 cells is
suppressed by a pathway that involves 53BP1 and RIF177,78 (Fig. 1).
Another event associated with DNA replication is the establish- 53BP1RIF1 and BRCA1CtIP are thus engaged in a battle for real
ment of sister chromatid cohesion. Its main effector, cohesin, promotes estate at the site of DNA ends that is refereed by CDKs and histone
HR in a variety of systems6870 and could be a key determinant for the modifications such as H4 (Lys16) and H2A (Lys15) acetylation89,90.
Sphase activation of HR. However, one must discriminate between the While this antagonism represents a mechanism regulating DSB
bias towards sister chromatid recombination caused by the tethering repair pathway choice during the cell cycle, recent evidence suggests
of chromatids versus choice between HR and competing pathways. that 53BP1 and BRCA1 may instead fine-tune each others activity.

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a
Unreplicated chromatin Replicated chromatin

MMS22L MMS22L MMS22L

TONSL TONSL TONSL

53BP1
Me Me Ub Me
53BP1
H4K20me2
MMS22L MMS22L

H4K20me0 Ub TONSL TONSL

NHEJ

sin
he
Co
HR

b G1 S G1 S
mid-S mid-S

M G2 M G2

P
Nu

Nu
cle

CDK activity low CDK sufficiently high HELB

cle
ar

CDK CDK

ar
en

HELB

en
vel

vel
ope

ope
CtIP

P
EXO1 RPA P
P
CtIP

M N M N
P

EXO1
R R

NHEJ HR

Figure 4 Models for cell-cycle-dependent activation of repair by homologous recombination. (a) The post-replicative chromatin marking model. Incorporation
of unmethylated histone H4 Lys20 in newly replicated chromatin. H4K20me0 is recognized by MMS22LTONSL, which promotes HR. H4K20me0 also
precludes binding of 53BP1. Cohesin also labels newly replicated chromatin and promotes HR. (b) The timer model. CDK activity progressively rises during
Sphase, triggering enough phosphorylation of NBS1, CtIP, HELB and EXO1 to stimulate resection and HR.

In particular, 53BP1 limits the extent of resection in S/G2 cells91, thereby
decreasing the probability of engaging mutagenic RAD52-dependent
SSA. Therefore, 53BP1 can be viewed, paradoxically, as a factor that
promotes the fidelity of HR91.
Risks and opportunities of genome scanning by the replisome
Semi-conservative DNA replication is a remarkable process that must
be precisely regulated to occur exactly once per cell cycle with mind-
boggling accuracy and speed especially considering that DNA rep-
lication forks must replicate past difficult DNA structures, repetitive
sequences, DNA lesions, RNADNA hybrids, tightly bound proteins
and collisions with transcribing RNA polymerases92. Alternatively, as
replisomes scan the entire genome, it also provides an opportunity for
the identification and repair of DNA lesions. Consequently, replica-
tion-dependent and -independent repair pathways exist for a subset
of lesions. A clear case of this situation is the repair of interstrand
crosslinks (ICLs). ICLs block replisome progression, which initiates
repair pathways that either involve the glycosylase NEIL3 (ref. 93)
or by the Fanconi anaemia (FA) group of proteins94. The replication-
dependent FA pathway coordinates incision on each side of the lesion,
translesion DNA synthesis followed by HR, and nucleotide excision
repair (NER), which together regenerate an undamaged DNA duplex.
While ICLs are a threat to genome replication, they also block tran-
scription and so their removal must occur independently of cell cycle
position. The block to RNA polymerase progression is a plausible
mechanism for the initiation of replication-independent ICL repair 95

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alongside the detection of helix-distorting ICLs by the NER pathway 96.
Replication-independent ICL repair is poorly understood, although it has
been shown in Xenopus laevis extracts to involve PCNA ubiquitylation
and DNA polymerase (ref.97) at a step downstream of lesion unhook-
ing 95. Exactly how replication-dependent and -independent ICL repair
pathways compete or cooperate to remove these toxic lesions is unknown.
Although ICL repair harnesses the genome-scanning properties
of DNA replication, the repair of most lesions at stalled forks is risky
because their excision in the context of an unwound template will result
in a DSB. To compensate for the inability of most types of excision repair
pathways to operate in ssDNA, cells have evolved the DNA damage tol-
erance pathway (also known as DNA damage bypass), which is not a
DNA repair process perse as it enable replisomes to bypass fork-blocking
lesions. This pathway consists of two general branches: template switch-
ing and translesion DNA synthesis, which are both initiated following
detection of extended stretches of RPA-coated ssDNA at stalled DNA
replication forks. The formation of ssDNA promotes various flavours
of PCNA ubiquitylation that activate either template switching or the
recruitment of translesion synthesis polymerases98. DNA damage toler-
ance is often referred to as post-replicative repair, and studies in budding
yeast have clearly established that it remains competent long after the
completion of bulk replication in G299,100 which suggests that daughter
strand gaps may be marked, perhaps by PCNA, until they are repaired.
M phase DNA repair deals with unfinished business
The segregation of chromatids to opposite poles is the core mission of
mitosis. It is therefore imperative that all links between sister chromatids
are severed at the onset of anaphase. While cohesin removal and sister
chromatid decatenation by topoisomeraseII clears most chromatid link-
ages101,102, DNA replication intermediates and other types of joint DNA
molecules such as HJs can persist until mitosis23,103. Mitotic cells, in fact,
activate pathways that process such joint molecules to promote chromo-
some segregation (Fig.3).
One such pathway is the mitotic activation of HJ resolution, which
is triggered by the CDK1-dependent phosphorylation of SLX4 and
EME1 at the onset of mitosis23. This event promotes the interaction of
SLX1SLX4, a HJ nickase, with MUS81EME1, which is active against
nicked HJs23. The other HJ resolvase, GEN1, is also activated upon
mitotic entry as it gains access to chromatin only after nuclear envelope
breakdown due to its nuclear export sequence104,105. Its budding yeast
orthologue, Yen1, is also excluded from the nucleus during interphase
due to phosphorylation that is reversed upon anaphase entry 106,107. As
HJ resolution results in the formation of either non-crossover or less
desirable crossover products, the mitotic activation of HJ resolution is
a last-ditch attempt to process the recombination intermediates that
escaped the attention of the BTR complex 108.
Late and incomplete DNA replication represents a second class of
liability for chromosome segregation. This is particularly true for loci
referred to as common fragile sites (CFSs) that are replicated late in the
cell cycle, especially under conditions in which replication is challenged
with low doses of the DNA polymerase inhibitor aphidicolin109,110. CFSs
are said to be expressed when metaphase chromosomes display gaps
or breaks110. CFSs usually reside in very large genes with long introns,
and their instability may arise from lack of DNA replication origins, late
DNA replication intermediates, DNA secondary structures, or collisions
between transcription and replication machineries111. CFS expression
depends on the action of endonucleases, such as MUS81EME1, in early
mitosis112,113. MUS81EME1 cleaves a broad range of branched DNA
structures23,114,115 that have been proposed to occur at CFSs, although the
exact nature of these structures is still under investigation.
In addition to processing late-replication intermediates, cells also
attempt to complete the replication of at-risk regions such as CFSs. In
particular, a POLD3-dependent DNA synthesis pathway, reminiscent
of break-induced replication, occurs at MUS81-processed CFSs in early
mitosis116. In the absence of MUS81EME1, cells display increased lev-
els of FANCD2-marked, DAPI-positive anaphase bridges along with
FANCD2-bound ultra-fine bridges (UFBs) that are DAPI-negative but
coated with the translocase PICH, the BTR complex and RIF1112,113,117119.
The localization of these proteins at CFSs may represent a last effort to
facilitate sister chromatid disjunction. Loss of these UFB binding and
processing factors leads to micronucleation and spontaneous 53BP1 foci
in the next G1 phase, which are often referred to as 53BP1 bodies. The
function of 53BP1 bodies is still mysterious, but they may mark, protect
or facilitate the repair of these inherited DNA lesions112,113. Collectively,
these findings indicate that CFS expression through the action of struc-
ture-specific nucleases in mitosis is beneficial for chromosome segrega-
tion rather than being a genome-destabilizing event.
Paradoxical inhibition of DSB repair in Mphase
While cells attempt to complete DSB repair before the onset of chromo-
some segregation, they might also face new DNA lesions during mitotic
progression. It has been known for decades that from late prophase
onwards, cells do not repair mitotic DSBs or delay mitotic progression
as a consequence of their detection120,121. There are some exceptions to
this situation: limited mitotic DNA repair occurs in response to etopo-
side treatment, perhaps reflecting the high activity of topoisomeraseII in
mitosis122,123, and a robust metaphase arrest can be induced by high doses
of DNA damage that impair centromere function, thereby activating the
spindle assembly checkpoint 124,125.
Mitotic cells do, however, sense DSBs, and can initiate a DSB signal-
ling cascade that is comparable, in its early stages, to that of interphase
cells. Indeed, NBS1 and Ku both localize to mitotic DSBs, and breaks
induce H2AX foci that colocalize with MDC1, as expected126132. H2AX
phosphorylation occurs in an ATM- and DNA-PK-dependent manner,
indicating that both kinases respond to mitotic DNA breaks128. However,
the signalling cascade downstream of MDC1 is interrupted in mitotic
cells, as RNF8, RNF168, BRCA1 and 53BP1 all fail to colocalize with
H2AX during this cell cycle stage128,129,133. The first block to this cascade
is at the level of RNF8 recruitment due to a CDK1-dependent inhibi-
tion of the RNF8MDC1 interaction134. The inhibitory CDK1 site on
human RNF8 is not conserved in other species, thus alternative means
to inhibit RNF8 recruitment in those species must exist. A second block
to this cascade involves 53BP1 phosphorylation in Mphase128,129,135,136
in particular, phosphorylation of the Thr1609 and Ser1618 residues
by CDK1 (or p38 kinase) and PLK1, respectively 134,136,137. The phospho-
rylation of these two residues inhibits the interaction of 53BP1 with
ubiquitylated nucleosomes67 and thus blocks its accumulation on dam-
aged chromatin134,136,137. Mutation of Thr1609 and Ser1618 to non-phos-
phorylatable residues alleviates the impediment to 53BP1 recruitment
and partially restores mitotic DSB repair (presumably through NHEJ).
The partial nature of mitotic DSB repair activation is likely to be due to
another block to mitotic NHEJ that involves XRCC4 phosphorylation122.

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 REVIEW

DNA repair stimulated by 53BP1 is, paradoxically, deleterious during ACKNOWLEDGEMENTS

the process of mitosis, as it leads to ionizing radiation hypersensitivity
and chromosome segregation defects that are caused by sister-telomere
fusions134. Mitotic telomeres often show signs of DNA damage, espe-
cially in cells undergoing prolonged mitotic arrest 138 or those that are
concurrently damaged with intrachromosomal DSBs134,139. Interestingly,
in both cases, the mitotic kinase AuroraB is critical for the induction
of a telomeric DNA damage response134,138. These observations suggest
that repair by NHEJ may be blocked in mitosis in order to tolerate some
levels of telomere uncapping, at the potential cost of mis-segregating
acentric fragments produced by mitotic DSBs on chromosome arms. The
function of this transient telomere uncapping is unknown but it could be
linked to telomere segregation or to a pathway that triggers the perma-
nent cell cycle arrest of damaged mitotic cells in the following G1 stage140.
While HJ resolution pathways are activated in mitosis, extensive
DNA end resection, CHK1 activation and RAD51 loading is blocked
in mitosis141,142. CHK1 inactivation is due to the degradation of Claspin,
its activator, at the onset of mitosis143. RAD51 loading is inhibited in
a CDK1-dependent manner, and ectopic CDK1 activation in Xenopus
Sphase extracts abrogates RAD51 chromatin binding 141. Both RPA and
BRCA2 are phosphorylated by cyclin-dependent kinases, which may
also contribute to the inhibition of RAD51 filament assembly or stabil-
ity 144146. Elevating CDK1 activity in interphase through WEE1 inhibition
decreases HR efficiency, emphasizing a role for CDK1 in blocking HR
in mitosis147.
Are mitotic chromosomal fragments held together?
Broken chromosomes represent a major problem for mitotic cells,
since the segregation of the resulting acentric chromatid fragments
should be random in the absence of compensatory mechanisms.
Such mechanisms would allow mitotic cells to suppress DNA repair
with minimal consequences. Evidence for their existence is found in
Drosophila melanogaster, in which the ends of mitotic DNA breaks are
tethered through a DAPI-stainable DNA bridge. This bridge is covered
with the mitotic kinases Polo, AuroraB and BubR1 and, importantly,
facilitates efficient segregation of acentric chromosome fragments148. It
is currently unclear whether a similar mechanism exists in mammals,
but it has been speculated that MRN could also carry out its DNA end-
tethering activity during mitosis149.
Conclusion
The cell division cycle is intimately linked with genome protection, and
this Review only shines light on a few salient examples. We anticipate
that the cell cycle regulation of DNA repair will continue to be a focus
of investigation for a number of reasons. Firstly, the development of
targeted cancer therapies that modulate cell cycle progression (CDK,
WEE1 and mitotic kinase inhibitors150) will impact the response to DNA
damage, and it may be possible to harness this regulatory intertwining
for therapeutic purposes. Secondly, the suppression of DNA end resec-
tion and HR in G1 (and G0) has profound implications for therapeutic
gene editing since it is currently impossible to undertake gene targeting
in non-dividing cells. Manipulating the mechanisms that suppress HR in
G1 cells may unlock the ability to precisely manipulate genomes in non-
dividing cells. We also expect that, in the coming years, the regulation of
other repair pathways by the cell cycle will be revealed, in particular how
the cell cycle modulates the various types of ICL repair.
We thank R. Szilard, M. Zimmermann, S. Noordermeer and A. Sartori for critical
reading of the manuscript, as well as D. DAmours and J. Lukas for stimulating
discussions and advice. N.H. is a long-term fellow of the Human Science Frontier
Program. D.D. is a Canada Research Chair (Tier1) in the Molecular Mechanisms
of Genome Integrity. Funding for work in the laboratory of D.D relating to the
regulation of DNA repair by the cell cycle include CIHR grant FDN143343 and a
Grant-in-Aid from the Krembil Foundation.
ADDITIONAL INFORMATION
Reprints and permissions information is available online at www.nature.com/
reprints. Correspondence should be addressed to D.D.
COMPETING FINANCIAL INTERESTS
N.H. declares no competing financial interests. D.D. is a paid advisor and
receives funding from CRISPR Therapeutics, and also receives funding from
Blueline Bioscience.
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