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# Recent advances in understanding DNA replication: cell type-specific adaptation of the DNA replication program

Antoine Aze, Domenico Maiorano

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Antoine Aze, Domenico Maiorano. Recent advances in understanding DNA replication: cell type-specific adaptation of the DNA replication program. F1000Research, 2018, 7, 10.12688/f1000research.15408.1. [hal-01887450](https://hal.science/hal-01887450)

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REVIEW

# Recent advances in understanding DNA replication: cell type-specific adaptation of the DNA replication program [version 1; peer review: 2 approved]

Antoine Aze, Domenico Maiorano

Institute of Human Genetics, UMR9002, CNRS-University of Montpellier, Montpellier, 34396 Cedex 5, France

v1

**First published:** 29 Aug 2018, 7(F1000 Faculty Rev):1351 (https://doi.org/10.12688/f1000research.15408.1)

**Latest published:** 29 Aug 2018, 7(F1000 Faculty Rev):1351 (https://doi.org/10.12688/f1000research.15408.1)

## Abstract

DNA replication is an essential process occurring prior to cell division. Cell division coupled to proliferation ensures the growth and renewal of a large variety of specialized cell types generated during embryonic development. Changes in the DNA replication program occur during development. Embryonic undifferentiated cells show a high replication rate and fast proliferation, whereas more differentiated cells are characterized by reduced DNA synthesis and a low proliferation rate. Hence, the DNA replication program must adapt to the specific features of cells committed to different fates. Recent findings on DNA synthesis regulation in different cell types open new perspectives for developing efficient and more adapted therapies to treat various diseases such as genetic diseases and cancer. This review will put the emphasis on recent progress made in this field.

## Keywords

DNA synthesis, nucleus, chromatin, epigenetics, development, cell cycle, differentiation

## Open Peer Review

### Reviewer Status

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### Invited Reviewers

1

2

version 1

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published

29 Aug 2018

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F1000 Faculty Reviews are written by members of the prestigious F1000 Faculty. They are commissioned and are peer reviewed before publication to ensure that the final, published version is comprehensive and accessible. The reviewers who approved the final version are listed with their names and affiliations.

Any comments on the article can be found at the end of the article.

<footnote n="1">1 **Christian Speck**, Institute of Clinical Science, Imperial College, London, UK</footnote>

<footnote n="2">2 **David MacAlpine**, Duke University Medical Center, Durham, USA</footnote>

<footnote>**Rachel Hoffman**, Duke University Medical Center, Durham, USA</footnote>
**Corresponding author:** Domenico Maiorano (domenico.maiorano@igh.cnrs.fr)

**Author roles:** Aze A: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing; Maiorano D: Supervision, Validation, Writing – Review & Editing

**Competing interests:** No competing interests were disclosed.

**Grant information:** Research in DM's laboratory is supported by grants from "Fondation ARC pour la Recherche sur le Cancer", La Ligue contre le Cancer, INSERM, and MSD Avenir.

*The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.*

**Copyright:** © 2018 Aze A and Maiorano D. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**How to cite this article:** Aze A and Maiorano D. Recent advances in understanding DNA replication: cell type-specific adaptation of the DNA replication program [version 1; peer review: 2 approved] F1000Research 2018, 7(F1000 Faculty Rev):1351 (https://doi.org/10.12688/f1000research.15408.1)

**First published:** 29 Aug 2018, 7(F1000 Faculty Rev):1351 (https://doi.org/10.12688/f1000research.15408.1)
**Introduction**

DNA synthesis occurs during the S phase of the cell cycle and is ensured by the replisome, a molecular machine made of a large number of proteins acting in a coordinated manner to synthesize DNA at many genomic locations, the replication origins¹. Replication origin activation in space and time (or replication program) is set by a sequence of events, starting already at the end of mitosis, lasting through G₁ phase, and ending in S phase when DNA replication is activated. These coordinated events ensure that the full genome will be replicated before mitosis since a faithful DNA synthesis is a prerequisite for genome integrity maintenance⁷. DNA replication initiates from thousands of replication origins scattered along the chromosomes. Origins acquire the competence to replicate in a step called “licensing” that involves formation of a pre-replication complex (pre-RC), including loading of the replicative helicase MCM2-7 (recently reviewed 3). Then once the transition from pre-RC to a pre-initiation complex (pre-IC) is induced by S-phase kinases, DNA replication is activated and DNA is unwound to provide the template for the replicative DNA polymerases²,⁴.

The last 30 years of deep investigations in the DNA replica-
tion field have allowed the general mechanisms involved in
eukaryotic DNA synthesis to be defined. Thanks to recent meth-
odological improvements, such as genome-wide analysis⁵,⁶,
in vitro reconstitution assays⁷⁻⁹, proteomics¹⁰⁻¹², and structural
biology¹³,¹⁴, our vision of the molecular pathways that govern
DNA synthesis is becoming sharper.

Although the factors that drive DNA replication initiation are
well conserved throughout the eukaryotic kingdom, cell type-
specific differences in the regulation of this process within
eukaryotes have been recently unraveled. From these studies,
it appears that the regulation of DNA replication is influenced
by the fate of a given cell. These findings shed new light on
how the DNA replication program and the proliferation rate are
being modulated during cell fate commitment, when cell type
specialization is determined. New discoveries in this topic are
undoubtedly essential to expand our understanding about the
genesis of diseases and their progression. In this review, we
will briefly describe recent achievements aiming to understand
how the replication factors involved in the licensing, activa-
tion, and elongation steps of DNA synthesis adapt dynamically
with cell fate determination to maintain genome integrity and
homeostasis.

Coordination between replication origin licensing and G₁ length is critical for cell destiny

*In vitro* reconstitution assays in yeast have provided tremendous detailed information on how origins are licensed by dissecting the sequential biochemical steps involved<sup>3,7-9,14,15</sup>. Detailed reviews covering the main features that characterize metazoan origins have been recently published<sup>3,5,16</sup>. Briefly, in eukaryotes, replication origins are licensed through a chronological order that requires the binding of Origin Recognition Complex (ORC), Cdc6, and Cdt1 proteins onto chromatin<sup>17,18</sup>. Two hexamers of the MCM2-7 helicase complex then are loaded in an inactive state prior to S phase (Figure 1). In yeast and mouse cells, Cdt1 and MCM2-7 form a complex before being recruited

onto replication origins by ORC and Cdc6<sup>19-21</sup>. The MCM2-7
double hexamer must be activated by S-phase kinases to be
able to unwind DNA and initiate DNA replication. However,
MCM2-7 complexes are recruited in excess so that not all
MCM2-7 complexes are activated during a cell cycle (Figure 1).
The choice of the origins to be activated (around 30,000 in
mammalian cells) is variable from cell to cell and ensures
flexibility in origin usage during the DNA replication pro-
gram to adapt with cell fate commitment, cell environment, and
replicative stress<sup>22,23</sup>. Origin mapping strategies coupled with
high-throughput sequencing revealed that the chromatin context
influences origin selection through genetic and epigenetic features
located in close proximity to the origins.

It is now clear that most replication origins being constitutively
activated across multiple metazoan cell types (hereafter named
"shared origins") are associated with unmethylated CpG islands,
G-rich elements, transcriptional start sites, and histone modi-
fications related to open chromatin marks (H3K4me3, H3K9Ac,
and H3K27Ac)²⁴⁻²⁹. Notably, this population of shared origins
tends to initiate early during S phase, whereas cell type-specific
origins initiate during late S-phase and are linked with
compacted chromatin marks²⁸. These observations underline the
relationship between chromatin modifications with the cellular
context in origin selection.

Two independent studies describing reconstitution of DNA
replication initiation events *in vitro* from chromatinized tem-
plates in yeast provided biochemical evidence that the regulatory
functions of chromatin structure influence origin selection. These
studies confirmed that nucleosome-free regions contribute to
defining ORC binding and thus origin function³⁰,³¹, as previously
suggested from genome-wide studies mapping ORC binding
sites and nucleosome occupancy in various organisms³²⁻³⁶.
Moreover, in mouse embryonic stem (ES) cells, depletion of
the histone H1 perturbs the landscape of replication origin
activation³⁷. Because chromatin environment changes during
cell fate commitment and in different cell types, it is generally
assumed that the DNA replication program is coordinated with
transcription to avoid transcription-replication conflicts and thus
preserve genomic integrity. This seems particularly crucial
during the onset of developmental programs and cell lineage
specification. In *Caenorhabditis elegans*, origins from rapidly
replicating pluripotent embryos coincide with open chromatin
regions of highly transcribed genes, whereas establishment
of the new transcriptional program, occurring at later embry-
onic stages when cell differentiation begins, correlates with the
reorganization of replication initiation sites²⁹,³⁸.

Permissive chromatin features for rapid activation of replication origins could influence the cell cycle length. ES cells have a shorter G₁ compared with their differentiated counterparts, which impacts on the licensing step. ES cells recruit much more MCM2-7 to the chromatin than tissue-specific stem cells or progenitor cells³⁹. This excess of origins that remain dormant during S phase appears to be important to maintain pluripotency⁴⁰,⁴¹ and could also protect the genome against DNA replication stress occurring in ES cells³⁹,⁴².
<figure><img src="image_7.png" /></figure>

**Figure 1. Replication of eukaryotic chromosomes.** Replication origins are scattered along the genome to ensure that each chromosome is entirely replicated in S phase. Pre-replication complex (Pre-RC) assembly and origin activation are tightly regulated in a sequential manner to ensure that replication occurs only once per cell cycle. Hence, throughout the G₁ phase, origins are licensed by the sequential loading of the pre-RC components: Origin Recognition Complex (ORC), CDC6, CDT1, and (at the final stage) MCM2-7. Once a double MCM2-7 hexamer is stably recruited onto chromatin, origins are licensed. Many replication origins are licensed but few are activated, allowing a backup of origins to be used when DNA replication is perturbed. Origin firing occurs under the combined activities of S-phase Cyclin-Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), allowing the recruitment of additional factors involved in DNA replication initiation and elongation. MCM2-7, together with CDC45 and GINS, composes the DNA helicase that unwinds DNA in a bidirectional manner. The entire replication machinery made of accessory factors required for replication fork stability and the synthesis of DNA is called the replisome.

Besides changes in the kinetics by which MCM2-7 is loaded onto chromatin, licensing control adapts to G₁ length, suggesting that pre-RC binding is developmentally regulated. Indeed, quantitative single-cell analysis performed in human cells by Matson and colleagues demonstrated that origins are licensed faster in pluripotent cells compared with their isogenic differentiated counterparts and that loading of the MCM2-7 helicase slows down as G₁ duration is extended⁴³. The authors revealed that high expression of the licensing factor Cdt1 was important for fast MCM2-7 loading rates, thus enabling ES cells to rapidly license origins prior to the G₁-to-S phase transition. Similar to the need for additional dormant origins, fast licensing kinetics seems essential to ensure pluripotency in ES cells and induced pluripotent stem cells. This observation was corroborated by Carroll and colleagues in intestinal stem cells from adult tissues⁴⁴. The authors demonstrated that licensing is interconnected with the proliferative commitment of stem cells. They found that Lgr5⁺ intestinal stem cells, which contribute primarily to the renewal of the intestinal epithelium, reside mostly in a G₁, unlicensed state, although they express MCM2 and other proliferative markers to a level equivalent to the licensed population. Interestingly, this unlicensed state correlates with an elongated cell cycle that could be considered as a backup mechanism to sustain proliferative fate decisions and tissue maintenance. Nonetheless, the link between chromatin structures and adaptation to G₁ length in such a context remains to be determined.

Uncoupling of G₁ length with licensing kinetics has been observed during oncogenic transformation. Overexpression of oncogenes, such as cyclin E and c-MYC, shortens G₁ and forces cells to engage S phase earlier with incomplete licensing. Consequently, the replication program is perturbed, leading to accumulation of replication stress⁴⁵,⁴⁶. Origin usage following oncogene activation was recently investigated genome-wide. The authors found that in these conditions new initiation zones, which were normally suppressed by transcription in G₁, appear in intragenic regions⁴⁷. These results also confirm an observation in *drosophila* showing that active transcription modulates MCM2-7 distribution⁴⁸. Nonetheless, how RNA polymerases inactivate and redistribute MCM2-7 remains to be determined. It has been reported that DNA translocases and RNA polymerases in yeast are able to push MCM2-7 along DNA, but similar processes have not yet been described in metazoans⁸,⁴⁹. An emerging picture from these studies is that cell cycle length, local chromatin structure, and active transcription can modulate the extent of origin licensing.

## Activation of DNA replication in different cellular contexts: control in space and time matters

During S phase, origins are activated by the combined activities of cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) kinases (Figure 1) targeting several substrates allowing the recruitment of CDC45 and GINS complex to
MCM2-7, thereby forming the CMG complex, the functional replicative helicase⁵⁰. Chromatin environment contributes to licensing as described previously through MCM2-7 loading but also to origin selection through MCM2-7 activation⁵¹,⁵². The dynamics of origin activation during S phase follow a spatiotemporal order known as replication timing (Figure 2). Replication timing in eukaryotes controls activation of replication of large chromosomal domains and is mediated by genetic determinants, local histone modifications, and global chromatin organization⁵³. Thus, DNA sequence features that mediate licensing in vertebrates can also affect the time when origins will be activated. Origin G-rich repeated elements (OGREs) are prone to form G-quadruplexes (G4s). Their presence in the genome is associated with origin activity²⁵,⁵⁴. Nucleosome organization at the proximity of origins was reported to modulate both origin licensing (as described above) and MCM2-7 helicase-dependent activation steps of initiation⁵⁵ in yeast and more recently in mammals³⁷. Different histone modifications and certain histone modifiers have been assigned in origin selection and the dynamics of their activation across S phase²⁹,³⁸,⁵²,⁵⁶⁻⁶⁰.

Several histone-modifying enzymes such as the methyltransferase PR-Set7 and the acetyltransferase HBO1 in complex with BRPF3 have been reported to have a role in activation of particular replication origins⁵²,⁶¹. More recently, a great deal of investigation has focused on the contribution of chromatin regulators in controlling the timing of activation through chromatin organization. The telomere-associated protein Rif1 interferes with DDK-dependent phosphorylation of MCM2-7 and modulates nuclear architecture by anchoring heterochromatin⁶²,⁶³. Rif1 might organize replication-timing domains through its association with G4s to repress pre-RC activation until late S-phase⁶³⁻⁶⁵. The ORC-associated protein ORCA/LRWD1 establishes a represistent chromatin environment at a subset of origins, thus priming them for late replication⁶⁶. The replication-initiation determinant protein RepID initiates replication in a sequence-specific manner by interacting with a subgroup of origins⁶⁷.

Modulation of origin activation in a cell type-specific manner depends upon global changes in chromosomal architecture and mainly occurs within large chromosome domains known

<figure><img src="image_8.png" /></figure>

**Figure 2. Replication program and cell features.** Changes in the DNA replication program are dependent on cell features. In those rapidly dividing embryos, which are transcriptionally inactive, many replication origins fire to ensure fast S-phase. Selection of replication origins at this stage is believed to be random. Despite improvements in replication origin mapping, this idea has not yet been challenged. The timing of replication during early development is not clearly defined, as S-phase length is very short. During the pluripotency stage, appearance of gap phases and the onset of transcription confer a certain nuclear organization that sets the genome for a specific program of replication. Constitutive origins that replicate early are associated with strong origin density and efficiency, a high GC content, strong gene density, and nucleosome-free regions. On the other side, late constitutive origins are poor in origins and genes, GC content is low, and chromatin accessibility is restricted. Rif1 is a major factor shown to modulate origin activity for late replicating domains. The replication program changes dynamically during differentiation or cell lineage development, in coordination with changes in transcriptional activity and chromatin organization. Whereas constitutive origins are activated at the same location and with the same timing, some origins will be regulated following acquisitions of new cellular features. Remarkably, tumor development leads to formation of a heterogeneous population of cells, including cells with stem-like properties, progenitors, and differentiated tumor cells. These cells have the ability to perpetuate their lineage, to give rise to differentiated cells, and to undergo rapid growth. These molecular features, recapitulated during early embryonic development, illustrate similarities between cancer stem cells and embryogenesis. Understanding the DNA replication program regulation in a cell type-specific manner will allow investigation of the processes behind tumorigenesis from a different perspective.
as Topologically Associating Domains (TADs)⁶⁸⁻⁷¹. These constitute structurally distinct chromatin domains covering several megabases where DNA interactions are favored. TADs show a strong correlation with replication timing domains in which several replication units are concomitantly activated. Nuclear reorganization that accompanies differentiation and the establishment of developmental programs result in changes in replication timing of the human genome⁷⁰,⁷²,⁷³; notably, alterations in replication timing have been detected in several diseases⁷⁴,⁷⁵.

Therefore, flexibility in origin activation seems critical to accom-
modate the dynamic changes in the transcriptional program
and genome repositioning (Figure 2). Indeed, cell type–specific
replication origins correlate with regions corresponding to differ-
entiation-specific and tissue-specific gene expression programs⁷⁶,⁷⁷.
Origins that are activated in a cell type–dependent manner
appear to replicate late in S phase²⁸. Histones and chromatin
modifications described above impose a crosstalk between repli-
cation and transcription and are interconnected during differen-
tiation and development²⁹,³⁸ although this relationship remains
enigmatic.

Origin activation dynamics that determine replication tim-
ing therefore might play a role in establishing local and glo-
bal chromatin structure to facilitate the cellular response to the
differentiation process. Remarkably, establishment of TADs
during early development in mammals requires DNA replication
but not transcription⁷⁸. Moreover, perturbations in DNA rep-
lication during development can cause epigenetic changes
(alleviation of repressive marks) potentially inherited by the next
generations⁷⁹.

Chromatin organization immediately following fertilization in vertebrates is particularly critical for DNA replication initiation and this occurs in a transcription-independent manner following fertilization⁸⁰.

The capacity to selectively modulate origin usage in a cell type-
specific manner suggests that the proteins involved in origin
activation might play specific functions. Indeed, replication ini-
tiation proteins, and their availability during S phase, can be
involved in dictating specificity in origin activation⁸¹⁻⁸⁵. The
Sld3 vertebrate homolog Treslin/TICRR, a CDK target that
acts as a binding site for TopBP1 and Mdm2 binding protein
MTBP (both proteins being required for GINS-CDC45
recruitment), was proposed to link chromatin acetylation to
DNA replication initiation efficiency and timing in different
cancer cell lines⁵⁸,⁸⁶. This observation shows that Rif1 is not
the only known site-specific regulator of DNA replication ini-
tiation and timing. Likewise, a function in origin efficiency has
been assigned to the Treslin partner MTBP, probably through
its ability to localize Treslin near G4 structures⁸⁷. Finally, Rif1
accessibility to chromatin was recently implicated in determining
the onset of late replication during embryonic development⁸⁸.

Overall, these studies suggest a functional interaction between
components of the replication machinery with chromatin modi-
fiers leading to reorganization of the genome architecture

during development. In turn, reorganization of the chromatin
architecture defines cell type–specific transcriptional programs
that feedback on the availability of replication proteins but
equally on the replication timing by shaping chromatin archi-
tecture in specific domains such as TADs. Hence, genetic
and epigenetic features set during developmental transitions
may function as selective ways to repress or activate licensing or
firing (or both) of a subset of origins, potentially within
replication timing domains. Precise origin mapping at the single-
molecule level in a single cell (using nanopore sequencing, for
instance) completed by *in vitro* reconstitution assays using human
proteins will undoubtedly bring to light new exciting concepts
in this field.

Adaptation of the replisome to fast proliferation

A large variety of cell types critical for tissue function are formed during early embryonic development, when cell commitment first takes place. The proliferative state decision is variable for each cell type and may influence cell cycle duration. Consequently, steps required for proliferation, such as genome duplication, must be tightly regulated with cell cycle progression to maintain homeostasis. For instance, embryonic cell division in metazoans exhibits dramatic lengthening of the cell cycle at the onset of gastrulation, a stage required for cell type specialisation and embryo patterning⁸⁹,⁹⁰. Lengthening of the total cell cycle time is achieved mostly by extension of the G₁ phase and moderately the S phase⁹¹.

A variation in replisome composition following DNA replica-
tion perturbations or between different cell types was unraveled
thanks to recent advances in quantitative proteomics at the
replication forks¹¹,⁹²,⁹³. Analysis of the replication machinery at the
forks by isolation of proteins on nascent DNA (iPOND) allows
comparison of protein abundance at replication forks in different
contexts. It was shown that replisomes of pluripotent stem
cells contain a particular protein network that accommodates
a high proliferative capacity with short cell cycle phases and
reduced endogenous DNA replication stress⁹⁴. Factors involved
in DNA repair, such as mismatch repair, but equally pluripotency
and epigenetic inheritance factors, such as the NuRD-HDAC
complex, were found to be enriched at replication forks.
Interestingly, independent studies confirmed the requirement of
a NuRD complex for DNA replication during early embryonic
development⁹⁵. ES cells seem to require additional factors at the
replication forks to cope with DNA replication perturbations.
Thus, Filia-Floped was identified by iPOND as a new protein
complex involved in resolution of stalled forks during a normal
S-phase in mouse ES cells compared with their differenti-
ated counterparts⁹⁶. It is likely that strategies set in stem cells
to ensure fast DNA replication may also be exploited in other
biological contexts requiring fast proliferation, including very
early embryogenesis and tumorigenesis. For example, the
Rad18 E3 ubiquitin ligase, a master regulator of translesion
DNA synthesis, is an abundant component of the DNA replica-
tion machinery during *Xenopus* early development and confers
to the replisome the ability to hijack DNA lesions and suppress
the DNA damage response, ensuring fast cell cycle progression.
Remarkably, Rad18 was found to be highly expressed in
glioblastoma cancer stem cells⁹⁷.
Overall, these recent observations converge toward the idea that replisome composition can be selected “*a la carte*” regarding the biological features set by specific cell types. Consistent with this idea, replication of specific genome locations like telomeres, common fragile sites, or centromeres has been shown to involve particular DNA replication partners at the vicinity of the DNA replication fork to ensure stability of those genomic regions⁹⁸⁻¹⁰⁰.

## Conclusions

A defective DNA replication program is the source of several pathologies during development (Meier-Gorlin syndrome) and during the adult life (carcinogenesis)⁴⁵,¹⁰¹. Metazoan origins lack consensus sequences and are associated with different structural features that confer cell type-specific replication. Additional mechanisms operate to fine-tune pre-RC assembly, origin activity, and replisome composition. This allows cells to coordinate a fast replication program with their fate, contributing to genome integrity maintenance despite DNA replication perturbations often observed during tumorigenesis and development. Identification of various factors involved in origin selection and their activity in a cell type-specific manner is a great source of interest. Such factors may often be deregulated during cancer development; thus, their targeting might constitute effective anti-cancer therapies.

Nevertheless, these discoveries are only the tip of the iceberg as limited information is currently available regarding their regulation. Biochemistry, electron cryomicroscopy analysis, *in vitro* reconstitution assays using vertebrate proteins, and genomic approaches on single cells should largely contribute to breakthrough findings in the field.

## Abbreviations

CDK, cyclin-dependent kinase; DDK, Dbf4-dependent kinase; ES, embryonic stem; G4, G-quadruplex; iPOND, isolation of proteins on nascent DNA; ORC, origin recognition complex; pre-RC, pre-replication complex; TAD, topological associated domain

## Grant information

Research in DM's laboratory is supported by grants from "Fondation ARC pour la Recherche sur le Cancer", La Ligue contre le Cancer, INSERM, and MSD Avenir.

*The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.*

## Acknowledgments

We thank Philippe Pasero and Eric Morency for critical reading of the manuscript.

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# Open Peer Review

Current Peer Review Status: ✓

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

## Editorial Note on the Review Process

F1000 Faculty Reviews are written by members of the prestigious F1000 Faculty. They are commissioned and are peer reviewed before publication to ensure that the final, published version is comprehensive and accessible. The reviewers who approved the final version are listed with their names and affiliations.

## The reviewers who approved this article are:

### Version 1

1 **David MacAlpine**

Department of Pharmacology and Cancer Biology, Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA, Durham, USA

**Rachel Hoffman**

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, USA

**Competing Interests:** No competing interests were disclosed.

2 **Christian Speck**

DNA Replication Group, Institute of Clinical Science, Imperial College, London, UK

**Competing Interests:** No competing interests were disclosed.

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