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\DOI{10.5802/crbiol.123}
\datereceived{2023-01-24}
\daterevised{2023-07-19}
\dateaccepted{2023-07-20}
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\begin{noXML}

\RubricEF{Review Article}{Article de revue}

\title{Impact of R-loops on oncogene-induced replication stress in
cancer cells}

\alttitle{Impact des R-loops sur le stress r\'{e}plicatif induit par
les oncog\`{e}nes dans les cellules canc\'{e}reuses}

\author{\firstname{Jonathan} \lastname{Heuz\'{e}}\CDRorcid{0009-0008-2870-521X}}
\address{Institut de G\'{e}n\'{e}tique Humaine, CNRS UMR 9002,
Universit\'{e} de Montpellier, 141~rue~de la Cardonille, 34396
Montpellier Cedex 5, France}
\email[J. Heuz\'{e}]{jonathan.heuze@igh.cnrs.fr}

\author{\firstname{Yea-Lih} \lastname{Lin}\CDRorcid{0000-0003-4063-0771}}
\addressSameAs{1}{Institut de G\'{e}n\'{e}tique Humaine, CNRS UMR 9002,
Universit\'{e} de Montpellier, 141 rue de la Cardonille, 34396
Montpellier Cedex 5, France}
\email[Y.-L. Lin]{yea-lih.lin@igh.cnrs.fr}

\author{\firstname{Armelle} \lastname{Lengronne}\CDRorcid{0000-0002-3140-7968}}
\addressSameAs{1}{Institut de G\'{e}n\'{e}tique Humaine, CNRS UMR 9002,
Universit\'{e} de Montpellier, 141 rue de la Cardonille, 34396
Montpellier Cedex 5, France}
\email[A. Lengronne]{armelle.lengronne@igh.cnrs.fr}

\author{\firstname{J\'{e}r\^{o}me} \lastname{Poli}\CDRorcid{0000-0001-8091-5507}}
\addressSameAs{1}{Institut de G\'{e}n\'{e}tique Humaine, CNRS UMR 9002,
Universit\'{e} de Montpellier, 141 rue de la Cardonille, 34396
Montpellier Cedex 5, France}
\email[J. Poli]{jerome.poli@igh.cnrs.fr}

\author{\firstname{Philippe} \lastname{Pasero}\CDRorcid{0000-0001-5891-0822}\IsCorresp}
\addressSameAs{1}{Institut de G\'{e}n\'{e}tique Humaine, CNRS UMR 9002,
Universit\'{e} de Montpellier, 141 rue de la Cardonille, 34396
Montpellier Cedex 5, France}
\email[P. Pasero]{philippe.pasero@igh.cnrs.fr}

\keywords{\kwd{Replication stress}\kwd{Transcription--replication
conflicts}\kwd{R-loops}\kwd{Genomic
instability}\kwd{Oncogenes}\kwd{Cancer}}

\altkeywords{\kwd{Stress r\'eplicatif}\kwd{Conflits
r\'eplication--transcription}\kwd{R-loops}\kwd{Instabilit\'e
g\'enomique}\kwd{Oncog\`enes}\kwd{Cancer}}

\shortrunauthors

\begin{abstract} 
Replication stress is an alteration in the progression of replication
forks caused by a variety of events of endogenous or exogenous origin.
In precancerous lesions, this stress is exacerbated by the deregulation
of oncogenic pathways, which notably disrupts the coordination between
replication and transcription, and leads to genetic instability and
cancer development. It is now well established that transcription can
interfere with genome replication in different ways, such as head-on
collisions between polymerases, accumulation of positive DNA supercoils
or formation of R-loops. These structures form during transcription
when nascent RNA reanneals with DNA behind the RNA polymerase, forming
a stable DNA:RNA hybrid. In this review, we discuss how these different
cotranscriptional processes disrupt the progression of replication
forks and how they contribute to genetic instability in cancer cells.
\end{abstract}

\begin{altabstract}
Le stress r\'eplicatif correspond \`a une alt\'eration de la
progression des fourches de r\'eplication caus\'e par une vari\'et\'e
d'\'ev\'enements d'origine endog\`ene ou exog\`ene. Dans les l\'esions
pr\'ecanc\'ereuses, ce stress est aggrav\'e par la d\'er\'egulation de
voies oncog\'eniques, qui perturbe notamment la coordination entre la
r\'eplication et la transcription du g\'enome et entraine une
instabilit\'e g\'en\'etique contribuant au d\'eveloppement du cancer.
Il est maintenant bien \'etabli que la transcription peut interf\'erer
avec la r\'eplication du g\'enome de diff\'erentes fa\c{c}ons, telles que
des collisions frontales entre polym\'erases, l'accumulation de
supertours positifs de l'ADN ou la formation de R-loops. Ces structures
se forment au cours de la transcription lorsque l'ARN naissant se
r\'eassocie avec l'ADN derri\`ere l'ARN polym\'erase, formant un
hybride ADN:ARN stable. Dans cette revue, nous discutons comment ces
diff\'erents processus cotranscriptionnels perturbent la progression
des fourches de r\'eplication et comment ils contribuent \`a
l'instabilit\'e g\'en\'etique des cellules canc\'ereuses.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}

Our genome is particularly vulnerable during the S phase of the cell
cycle. During this process, the two strands of the double helix are
separated and the complementary strands are faithfully synthesized so
that the mother cell can pass two identical copies of the genome to the
daughter cells. This task is performed by thousands of molecular
micromachines called replisomes, which polymerize DNA at structures
called replication forks~\cite{1,2}. This process is \mbox{initiated} at
well-defined genetic elements called replication origins~\cite{3}. It
depends on the assembly of pre-replication complexes on origins during
the G$_{1}$ phase of the cell cycle and their activation in S phase
under the control of CDK and DDK kinases~\cite{4,5,6}. Origin
activation allows the recruitment of the CMG helicase complex,
consisting of the CDC45 protein, the MCM2-7 hexamer and the GINS
tetrameric complex. The CMG helicase interacts with the Fork Protection
Complex (composed of Claspin, Timeless, Tipin), as well as DNA
polymerases, and various accessory \mbox{factors} of the
replisome~\cite{1,7,8}. \mbox{Replication} origins are activated sequentially
throughout the S phase, following a precise spatiotemporal
program~\cite{9,10}. The replication forks progress at a rate of
1--2~kb per minute along the chromosomes until they encounter forks
progressing in the opposite direction or reach the end of the
chromosomes~\cite{11}. The two replisomes are then disassembled in a
process called replication termination~\cite{12}. Correct execution of
the replication program allows each portion of the genome to be
replicated once and only once during the S phase and ensures faithful
transmission of genetic material to daughter cells. However, the
replication forks encounter various obstacles during their progression
along the chromosomes, causing what is commonly referred to as
replication stress~\cite{13}.

\section{Mechanisms of response to replication stress}

Blocking replication forks can induce chromosomal breaks and
rearrangements. In fact, replication defects represent the major source
of endogenous genomic instability at the origin of cancers. Stalled
forks are unstable structures that must be rapidly stabilized and
restarted to avoid the formation of toxic recombination intermediates
and the induction of chromosomal rearrangements~\cite{11}. During
evolution, complex surveillance mechanisms have emerged in eukaryotic
cells, the main one being the S~phase checkpoint mediated by the
proteins ATR and CHK1~\cite{14}.

The ATR kinase is recruited to the forks via its ATRIP subunit, which
interacts with RPA, a complex that protects single-stranded DNA. ATR is
activated by TopBP1, a factor recruited to junctions between single-
and double-stranded DNA~\cite{15}. Once activated, ATR phosphorylates
the effector kinase CHK1, which allows the amplification and
propagation of the stress signal. CHK1 activation is mediated by the
Claspin protein, which is part of the replication fork protection
complex~\cite{16,17,18}. The ATR-CHK1 pathway acts at multiple levels to
coordinate fork repair processes, prevent premature entry into mitosis,
and enable completion of DNA replication~\cite{14}.

Fork restart depends on the concerted action of numerous enzymes
including homologous recombination factors, helicases, nucleases,
translocases and chromatin remodelers~\cite{19}. Under replication
stress conditions, these factors ensure extensive \mbox{remodeling} of the
fork structure. Specifically, this involves a remodeling of nascent DNA
strands to form a four-way junction called a reversed
fork~\cite{20,21}.  Fork reversal allows for the controlled degradation
of nascent DNA strands by the nucleases MRE11, DNA2, and EXO1~\cite{22}
and the recruitment of the recombinase RAD51 to initiate fork repair by
homologous recombination~\cite{23}. Excessive nascent strand resection
is prevented by BRCA1 and BRCA2, two repair proteins frequently mutated
in breast and ovarian cancers~\cite{24,25}. The inability of cells to
respond effectively to replication stress results in chronic genomic
instability and contributes to tumorigenesis.

\section{Main sources of replication stress}

Cellular responses to replication stress have been particularly studied
in the presence of drugs such as hydroxyurea (HU) or aphidicolin, which
block replication by inducing nucleotide pool depletion or inhibiting
DNA polymerase activity, respectively. Replication stress is also
induced by most drugs commonly used in chemotherapy, such as alkylating
agents, antimetabolites and topoisomerase inhibitors~\cite{26}. These
drugs induce uncoupling between helicase and polymerase activities and
increase single-stranded DNA, which is recognized as a universal stress
signal by the ATR kinase~\cite{13,27}. Interestingly, recent evidence
indicates that HU can also slow down fork progression independently of
dNTP pools by inducing oxidative stress and displacing Timeless from
the replisome~\cite{28,29}.

Under normal growth conditions, cells are also exposed to endogenous
sources of replication stress. This stress can originate from protein
complexes strongly associated with DNA, secondary structures of DNA
(G-quadruplexes, hairpins, etc.), or lesions induced by cellular
metabolism byproducts~\cite{13,19}. These obstacles are generally well
managed by the cell and their impact on replication remains limited. A
more complex problem concerns the availability of deoxyribonucleotides
(dNTPs) during S~phase. Indeed, the production of dNTPs must be closely
coordinated with DNA synthesis to ensure optimal progression of
replication forks~\cite{11}. Thus, it has been shown that a deficit of
dNTPs slows down the forks and an excess increases their
speed~\cite{30}. A too large or unbalanced pool of dNTPs can also
increase the frequency of mutations. Maintaining a balanced pool of
dNTPs throughout S~phase requires a tight coupling between the cell
cycle and cell metabolism~\cite{31}. Thus, cells must be able to detect
ongoing replication in order to produce dNTPs at the right time. Recent
evidence indicates that this is an essential function of the ATR-CHK1
pathway~\cite{32}. In addition, ribonucleotides (rNTPs) can be
mistakenly incorporated during DNA synthesis. These ribonucleotides are
normally removed by RNase H2~\cite{33,34,35}. In the absence of RNase~H2,
excision of ribonucleotides by topoisomerase~I (Top1) generates
non-repairable DNA lesions leading to deletions and double-strand
breaks~\cite{36}.

Another major source of endogenous replication stress results from
conflicts between replication and transcription. This is due to the
fact that DNA and RNA polymerases must share the same DNA substrate as
they move along the chromosomes, making conflicts
inevitable~\cite{37}. These conflicts can manifest in different ways,
such as head-on collisions between the transcription and replication
machineries, accumulation of topological stress along the DNA or the
formation of R-loops~\cite{38}. R-loops are three-stranded nucleic acid
structures that form during transcription when nascent RNA reanneals
with the template DNA strand, leaving the non-coding strand
unpaired~\cite{39,40}. Recent evidence indicates that R-loops
represent barriers to replication, but the molecular mechanisms
involved in this interference are still poorly understood~\cite{38}.

In the absence of exogenous sources of replication stress
(Figure~\ref{fig1}A), the low level of endogenous replication stress is
properly managed by the cell. Indeed, it enters S~phase only when its
metabolic state allows it, regulates dNTPs pools and coordinates
transcription and replication programs to limit conflicts~\cite{11}. In
contrast, aberrant activation of oncogenes in pre-tumor cells disrupts
this coordination (Figure~\ref{fig1}B). Endogenous replication stress
increases, which induces genetic instability and contributes to cancer
development~\cite{41}.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Major sources of endogenous replication stress in
normal and cancer cells. (A) Replication fork progression depends on a
constant supply of deoxyribonucleotide triphosphate (dNTPs) to fuel the
activity of DNA polymerases $\upalpha$, $\updelta$ and $\upvarepsilon$. The
cell must also avoid collisions between replication forks and
transcription complexes (RNAPII). Under normal growth conditions, this
coordination is achieved by tight coupling between replication and
transcription programs, cell cycle progression, and cell metabolism.
(B) Deregulation of oncogenic pathways increases transcription and
R-loop formation, alters the G$_{1}$/S transition, and disrupts cell
metabolism. This leads to deregulation of dNTP pools, increased
replication--transcription conflicts and accumulation of chromosomal
rearrangements.}
\end{figure*}

\section{Oncogene-induced replication stress}

It has been shown that aberrant expression of oncogenes such as Ras,
Myc, or CycE can disrupt DNA replication in a variety of ways, for
example by deregulating dNTP pools, increasing
replication--transcription conflicts, or causing premature entry into S
phase~\cite{39,41,42,43}. This oncogene-induced replication stress
represents a double-edged sword for cancer cells. While it contributes
to cancer development by promoting genetic instability, it also slows
tumor cell proliferation and activates anticancer barriers leading to
apoptosis or senescence~\cite{44,45,46,47}. To continue to proliferate,
cancer cells must circumvent these barriers, while limiting the
deleterious effects of replication stress on DNA replication. To do so,
cancer cells become very dependent on the ATR-CHK1
pathway~\cite{48,49,50}. They also adapt to oncogene-induced
replication stress by overexpressing components of the replication fork
protection complex such as Claspin and Timeless, independently of ATR
signaling~\cite{51}. This overexpression is observed in primary colon,
breast and lung carcinomas and is generally associated with poor
prognosis. Surprisingly, overexpression of Claspin and Timeless appears
spontaneously in immortalized human fibroblasts expressing the
H-Ras$^{\mathrm{V}12}$ oncogene and allows them to escape replication
stress and senescence~\cite{51}.

\section{Replication--transcription conflicts and cancer}

Recent evidence indicates that the replication stress detected in
cancer cells is largely due to replication--transcription
conflicts~\cite{43}. Since transcription and replication complexes
progress at roughly the same rate, the most severe conflicts result
from head-on collisions~\cite{52,53}. In order to limit the deleterious
effects of head-on conflicts, the genome of bacterial species such as
\textit{Bacillus subtilis} has evolved such that most genes are
codirectionally oriented with respect to replication forks~\cite{54}.
In contrast, genes associated with pathogenesis and stress resistance
are oriented in the opposite direction to replication, which locally
increases DNA damage and promotes targeted evolution of these
genes~\cite{55,56}. This codirectional organization of replication and
transcription is not found in eukaryotes because unlike bacterial
chromosomes, which have only one origin of replication, eukaryotic
chromosomes can carry several thousand. Moreover, the diversity of gene
expression programs in metazoans does not allow to define a unique
organization that would be adapted to all cell types. However,
eukaryotes are not helpless against transcription--replication
conflicts. Indeed, in the dynamic context of cell differentiation,
human cells use a trick of promoting replication initiation upstream of
highly transcribed genes~\cite{57,58,59}, allowing these genes to be
mostly replicated codirectionally. Thus, the average fork direction
profile is positive (codirectional) between the transcriptional start
(TSS) and termination (TTS) site in HeLa cells (Figure~\ref{fig2}A).
Interestingly, the direction of fork progression reverses at the end of
the genes, indicating that replication forks initiated downstream of
the genes converge with transcription at the TTS~\cite{60}.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Functional organization of the human genome and
replication--transcription conflicts. (A) Distribution of replication
fork direction (blue), R-loops (red) and a replication stress marker
(p-RPA32-S33, black) at a metagene (16,000 human genes) aligned between
transcription start (TSS) and termination (TTS) sites. A positive
replication fork direction indicates that the fork is progressing in
the same direction as transcription. (B) Schematic representation of
the distribution of replication origins and R-loops~(1), as well as RNA
polymerases prior to replication (2), when replication is initiated
upstream of the TSS~(3), and when replication and transcription
converge at the TTS, inducing replication stress (yellow spark) and
accumulation of phospho-RPA32-S33~(4).}
{\vspace*{1pt}}
\end{figure*}

Another important question concerns the impact of R-loops on
replication--transcription conflicts. Using methods such as DRIP-seq,
which involve immunoprecipitating R-loops with an antibody specifically
recognizing DNA:RNA hybrids and sequencing the associated nucleic
acids, it has been shown that R-loops are highly abundant and can
occupy up to 5\% of the human genome~\cite{61,62}. These structures
play important physiological roles, such as control of gene expression,
recombination of immunoglobulin genes, and mitochondrial DNA
replication~\cite{39}. The question then arises as to whether all
R-loops are inherently difficult to replicate, or whether a particular
class of R-loops is particularly toxic to replication forks.

Analysis of the distribution of replication stress markers such as the
ATR substrate phospho-RPA32-S33 showed that most R-loops are not
associated with replication stress in the human genome~\cite{60}.
Indeed, cotranscriptional R-loops are mainly located at the
transcription initiation (TSS) and termination (TTS) sites, whereas an
enrichment of phospho-RPA32-S33 is only detected at TTS
(Figure~\ref{fig2}A). Since these regions are on average replicated in
the opposite direction to the direction of transcription, this means
that replication--transcription conflicts occur preferentially at the
TTS of highly transcribed genes enriched in R-loops, when the
replication and transcription machineries converge
(Figure~\ref{fig2}B).  Importantly, these conflicts do not
automatically induce chromosomal breakage. Indeed, these events are
only detected in Top1-depleted cells, which fail to manage the
topological problems induced by replication and transcription
convergence (Figure~\ref{fig3}A).  Furthermore, the replication stress
observed in the absence of Top1 is suppressed by overexpression of
RNase~H1, an enzyme involved in the degradation of DNA:RNA hybrids,
attesting to the fact that R-loops contribute to
replication--transcription conflicts~\cite{60,63}.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Model of replication--transcription conflict
involving the formation of post-replicative RNA:DNA hybrids. (A)
Convergence of the replisome and RNA polymerases (RNAPII) at
transcription termination sites (TTS) induces accumulation of positive
DNA supercoiling, which causes replication fork arrest and activation
of the ATR kinase. The release of this topological stress is mediated
by Top1. (B) ATR promotes the removal of fork-blocking RNA polymerases.
At the same time, the DNA:RNA hybrid is degraded by RNase H to remove
the R-loop. (C) In the absence of RNase H, the replicative helicase
progresses beyond the position of the R-loop, transferring the DNA:RNA
hybrid behind the fork and stops at the next RNAPII. (D) RNA:DNA
hybrids located behind the fork interfere with post-replicative
mechanisms such as resection of nascent DNA and prevent fork restart.}
\end{figure*}

In conclusion, the functional organization of the human genome reduces
the genetic instability associated with replication--transcription
conflicts by limiting these conflicts to well-defined areas downstream
of the genes. However, this regulation cannot operate if the initiation
of replication takes place inside the genes, which occurs only very
rarely under normal physiological conditions. Interestingly, a new
class of replication origins has been identified within genes under
oncogenic stress~\cite{64}. This intragenic initiation is associated
with chromosomal rearrangements in cancers, possibly because it
prevents cells from handling replication--transcription conflicts in an
optimal manner.

\section{R-loops and post-replicative DNA:RNA hybrids}

The cell is well equipped to manage conflicts between replication and
transcription. On the one hand, the replication fork is able to
displace transcription complexes that block its passage
(Figure~\ref{fig3}B) by mobilizing various factors coordinated by the
ATR kinase~\cite{65,66,67,68}. On the other hand, the cell has a whole
array of enzymes capable of degrading or displacing DNA:RNA
hybrids~\cite{40}. These include RNases~H1 and H2, which specifically
degrade the RNA portion of DNA:RNA hybrids~\cite{69,70,71}. RNase~H2 is
frequently mutated in a severe interferonopathy called
Aicardi--Gouti\`{e}res syndrome~\cite{72,73}. Other key players include
helicases such as Senataxin~\cite{74,75} and FANCM~\cite{76}. Yet,
despite this redundancy of repair mechanisms, some R-loops interfere
with replication, following a mechanism that remains to be elucidated.

It is generally accepted that DNA:RNA hybrids are inherently difficult
to replicate and block fork progression. However, when replication and
transcription converge, the DNA:RNA hybrid is positioned on the strand
opposite the replicative helicase (CMG), which should not block its
progression (Figure~\ref{fig3}C). Recent data derived from an
\textit{in~vitro} replication system indicate that DNA:RNA hybrids do
not block forks in this configuration, unless the displaced DNA strand
is capable of forming a G4-like secondary structure~\cite{77}.
Alternatively, DNA:RNA hybrids could interfere with post-replicative
mechanisms acting behind the forks to promote their
restart~\cite{78,79}.  This model is supported by unpublished data from
our team, showing that in the absence of RNase~H2, the resection of
nascent DNA strands is inhibited by the persistence of undegraded
DNA:RNA hybrids. Since fork resection contributes to fork
restart~\cite{23,80}, these post-replicative DNA:RNA hybrids could
induce replication stress not only by blocking forks, but also by
preventing their restart (Figure~\ref{fig3}D). This model is in
agreement with recent data from the team of Massimo Lopes~\cite{81},
demonstrating by electron microscopy the presence of DNA:RNA hybrids
behind the replication forks.

These recent findings led us to propose a model in which R-loops and
transcriptional complexes would not only represent an obstacle to the
replication fork, but that the limited progression of replicative
helicase at the site of conflict could convert the R-loop into a toxic
DNA:RNA hybrid, interfering with fork restart if not rapidly degraded
by RNase~H2 (Figures~\ref{fig3}C,D). This model emphasizes the
importance for the cell of coordinated removal of the transcription
complex and the R-loop to avoid the formation of a post-replicative
DNA:RNA hybrid. It is worth noting that cells could be particularly
sensitive to transcription--replication conflicts when replication
forks are slowed by HU or oxidative stress~\cite{29} and that different
mechanisms may operate when fork progression is blocked. Indeed, it has
been reported that fork recovery at lesions caused by the Top1 poison
camptothecin (CPT) involves the MUS81-dependent cleavage of stalled
forks in human cells~\cite{82}. In fission yeast, fork restart at CPT
lesions and at the \textit{RTS1} replication fork barrier depends on
the degradation by RNase~H of RNA primers on Okazaki
fragments~\cite{83}.

\section{Conclusion and perspectives}

In conclusion, the data gathered by different groups in the last few
years shed new light on the molecular mechanisms at the origin of
genetic instability in cancers. It now appears that despite optimal
functional organization of the genome and redundancy of repair
mechanisms, replication--transcription conflicts represent a major
source of genetic instability, especially when oncogenic pathways are
deregulated. R-loops contribute significantly to these conflicts, but
the molecular mechanisms involved are more complex than initially
thought. Indeed, these structures would not only act as replication
barriers, but also act as postreplicative RNA:DNA hybrids to interfere
with the restart of stalled forks. Such postreplicative RNA:DNA hybrids
have recently been imaged by electron microscopy~\cite{81}, but the
mechanism of their formation remains unclear. New technologies such as
nanopore sequencing could help determine the relative position of
nascent DNA and RNA:DNA hybrids at the single molecule level. Another
important question that remains to be elucidated is whether these
post-replicative hybrids pre-exist before fork passage, as shown in
Figure~\ref{fig3}, or whether they are synthesized \textit{de~novo} by
RNA polymerases acting behind the stalled fork. From this point of
view, it is interesting to note that DNA:RNA hybrids are also detected
at DNA double-strand breaks and that these structures interfere with
DNA end resection, thus affecting homologous recombination repair
mechanisms~\cite{84}. Finally, it is now well established that the
repair mechanisms of forks and chromosomal breaks generate small DNA
fragments that accumulate in the cytoplasm, activate the cGAS-STING
pathway, and induce an inflammatory response~\cite{85,86}. A very
recent study also shows that DNA:RNA hybrids from R-loops after
cleavage by repair enzymes can also activate the cGAS-STING
pathway~\cite{87}. These data indicate that replication stress
generated by replication--transcription conflicts and R-loops induces
cellular responses acting beyond the boundaries of the cell. This
endogenous stress could thus stimulate the elimination of abnormal
cells by the immune system, opening promising prospects for the
development of new cancer\break therapies.

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgements}

The author thanks the members of the team ``Maintenance of genome
integrity during replication'' for their contribution to the work
mentioned in this review. Our work is supported by the Agence Nationale
de la Recherche (ANR), the Institut National du Cancer (INCa), the
Ligue contre le Cancer (labelled team) and the MSDAvenir Fund.

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