Rif1 restrains the rate of replication origin firing in Xenopus laevis.

Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles. Here, we find that in the absence of Rif1, patterns of replication foci change along with the acceleration of replication cluster activation. However, initiations increase only moderately inside active clusters. Our numerical simulations suggest that the absence of Rif1 compresses the temporal program towards more homogeneity and increases the availability of limiting initiation factors. We experimentally demonstrate that Rif1 depletion increases the chromatin-binding of the S phase kinase Cdc7/Drf1, the firing factors Treslin, MTBP, Cdc45, RecQL4, and the phosphorylation of both Treslin and MTBP. We show that Rif1 globally, but not locally, restrains the replication program in early embryos, possibly by inhibiting or excluding replication factors from chromatin.

A non-transcriptional function of Yap regulates the DNA replication program in Xenopus laevis.

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

Polo-like kinase 1 (Plk1) regulates DNA replication origin firing and interacts with Rif1 in Xenopus.

The activation of eukaryotic DNA replication origins needs to be strictly controlled at multiple steps in order to faithfully duplicate the genome and to maintain its stability. How the checkpoint recovery and adaptation protein Polo-like kinase 1 (Plk1) regulates the firing of replication origins during non-challenged S phase remained an open question. Using DNA fiber analysis, we show that immunodepletion of Plk1 in the Xenopus in vitro system decreases replication fork density and initiation frequency. Numerical analyses suggest that Plk1 reduces the overall probability and synchrony of origin firing. We used quantitative chromatin proteomics and co-immunoprecipitations to demonstrate that Plk1 interacts with firing factors MTBP/Treslin/TopBP1 as well as with Rif1, a known regulator of replication timing. Phosphopeptide analysis by LC/MS/MS shows that the C-terminal domain of Rif1, which is necessary for its repressive action on origins through protein phosphatase 1 (PP1), can be phosphorylated in vitro by Plk1 on S2058 in its PP1 binding site. The phosphomimetic S2058D mutant interrupts the Rif1-PP1 interaction and modulates DNA replication. Collectively, our study provides molecular insights into how Plk1 regulates the spatio-temporal replication program and suggests that Plk1 controls origin activation at the level of large chromatin domains in vertebrates.

Organization of DNA Replication Origin Firing in Xenopus Egg Extracts: The Role of Intra-S Checkpoint.

During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: (1) a segmentation of the genome into regions of low and high probability of origin firing; (2) that regions with high probability of origin firing escape intra-S checkpoint regulation and (3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering.

Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the Xenopus in vitro system.

Polo-like kinase 1 (Plk1) is a cell cycle kinase essential for mitosis progression, but also important for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is expressed in S phase, little is known about its function during unperturbed DNA replication. Using Xenopus laevis egg extracts, mimicking early embryonic replication, we demonstrate that Plk1 is simultaneously recruited to chromatin with pre-replication proteins where it accumulates throughout S phase. Further, we found that chromatin-bound Plk1 is phosphorylated on its activating site T201, which appears to be sensitive to dephosphorylation by protein phosphatase 2A. Extracts immunodepleted of Plk1 showed a decrease in DNA replication, rescued by wild type recombinant Plk1. Inversely, modest Plk1 overexpression accelerated DNA replication. Plk1 depletion led to an increase in Chk1 phosphorylation and to a decrease in Cdk2 activity, which strongly suggests that Plk1 could inhibit the ATR/Chk1-dependent intra-S phase checkpoint during normal S phase. In addition, we observed that phosphorylated Plk1 levels are high during the rapid, early cell cycles of Xenopus development but decrease after the mid-blastula transition when the cell cycle and the replication program slow down along with more active checkpoints. These data shed new light on the role of Plk1 as a positive regulating factor for DNA replication in early, rapidly dividing embryos.

Genome wide decrease of DNA replication eye density at the midblastula transition of Xenopus laevis.

During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a Xenopus laevis embryonic in vitro replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density. Increasing the dNTP pool did not accelerate S phase or increase replication eye density implying that dNTPs are not rate limiting for DNA replication at the Xenopus MBT. Increasing the ratio of DNA:cytoplasm in egg extracts faithfully recapitulates changes in the spatial replication program in embryos, supporting the hypothesis that titration of soluble limiting factors could explain the observed changes in the DNA replication program at the MBT in Xenopus laevis.

On the Interplay of the DNA Replication Program and the Intra-S Phase Checkpoint Pathway.

DNA replication in eukaryotes is achieved by the activation of multiple replication origins which needs to be precisely coordinated in space and time. This spatio-temporal replication program is regulated by many factors to maintain genome stability, which is frequently threatened through stresses of exogenous or endogenous origin. Intra-S phase checkpoints monitor the integrity of DNA synthesis and are activated when replication forks are stalled. Their activation leads to the stabilization of forks, to the delay of the replication program by the inhibition of late firing origins, and the delay of G2/M phase entry. In some cell cycles during early development these mechanisms are less efficient in order to allow rapid cell divisions. In this article, we will review our current knowledge of how the intra-S phase checkpoint regulates the replication program in budding yeast and metazoan models, including early embryos with rapid S phases. We sum up current models on how the checkpoint can inhibit origin firing in some genomic regions, but allow dormant origin activation in other regions. Finally, we discuss how numerical and theoretical models can be used to connect the multiple different actors into a global process and to extract general rules.

Tight Chk1 Levels Control Replication Cluster Activation in Xenopus.

DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data.

ATR/Chk1 pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells.

DNA polymerase eta (poleta) performs translesion synthesis past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XP-V) syndrome. The slight sensitivity of XP-V cells to UV is dramatically enhanced by low concentrations of caffeine. So far, the biological explanation for this feature remains elusive. Using DNA combing, we showed that translesion synthesis defect leads to a strong reduction in the number of active replication forks and a high proportion of stalled forks in human cells, which contrasts with budding yeast. Moreover, extensive regions of single-strand DNA are formed during replication in irradiated XP-V cells, leading to an over-activation of ATR/Chk1 pathway after low UVC doses. Addition of a low concentration of caffeine post-irradiation, although inefficient to restore S-phase progression, significantly decreases Chk1 activation and abrogates DNA synthesis in XP-V cells. While inhibition of Chk1 activity by UCN-01 prevents UVC-induced S-phase delay in wild-type cells, it aggravates replication defect in XP-V cells by increasing fork stalling. Consequently, UCN-01 sensitizes XP-V cells to UVC as caffeine does. Our findings indicate that poleta acts at stalled forks to resume their progression, preventing the requirement for efficient replication checkpoint after low UVC doses. In the absence of poleta, Chk1 kinase becomes essential for replication resumption by alternative pathways, via fork stabilization.

Y RNA functions at the initiation step of mammalian chromosomal DNA replication.

Non-coding Y RNAs have recently been identified as essential novel factors for chromosomal DNA replication in mammalian cell nuclei, but mechanistic details of their function have not been defined. Here, we identify the execution point for Y RNA function during chromosomal DNA replication in a mammalian cell-free system. We determined the effect of degradation of Y3 RNA on replication origin activation and on fork progression rates at single-molecule resolution by DNA combing and nascent-strand analysis. Degradation of Y3 RNA inhibits the establishment of new DNA replication forks at the G1- to S-phase transition and during S phase. This inhibition is negated by addition of exogenous Y1 RNA. By contrast, progression rates of DNA replication forks are not affected by degradation of Y3 RNA or supplementation with exogenous Y1 RNA. These data indicate that Y RNAs are required for the establishment, but not for the elongation, of chromosomal DNA replication forks in mammalian cell nuclei. We conclude that the execution point for non-coding Y RNA function is the activation of chromosomal DNA replication origins.

Use of DNA combing to study DNA replication in Xenopus and human cell-free systems.

The Xenopus egg extract has become the gold standard for in vitro studies of metazoan DNA replication. We have used this system to study the mechanisms that ensure rapid and complete DNA replication despite random initiation during Xenopus early development. To this end we adapted the DNA combing technique to investigate the distribution of replication bubbles along single DNA molecules. DNA replicating in egg extracts is labelled by addition of digoxigenin-11-dUTP and/or biotin-16-dUTP at precise times. These two dTTP analogues are efficiently incorporated into DNA during replication in the extract. After DNA purification and combing the DNA is visualized with appropriate fluorescent antibody/streptavidin molecules. Replicated DNA appears as green or red tracts whose pattern reveals how each molecule was replicated, allowing to follow the dynamics of DNA replication through S phase. We describe (a) the preparation and use of egg extracts and demembranated sperm chromatin templates; (b) a simple method for preparing silanized glass coverslips suitable for DNA combing and fluorescence detection; (c) two alternative replicative DNA labelling schemes and their respective advantages; and (d) a protocol for combining replicative labelling with detection of specific DNA sequences by fluorescent in situ hybridization (FISH). Although most observations made in Xenopus egg extracts are applicable to other eukaryotes, there are differences in cell-cycle regulation between mammalian somatic cells and embryonic amphibian cells, which led to the development of human cell-free systems that can initiate semi-conservative chromosomal DNA replication under cell-cycle control. We have employed the knowledge gained with Xenopus extracts to characterize DNA replication intermediates generated in human cell-free systems using DNA combing. We describe here (a) the preparation and use of human cell-free extracts and initiation-competent template nuclei for DNA combing studies; (b) an optimized labelling scheme for DNA replication intermediates by molecular combing and fluorescence microscopy.

DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts.

Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to progressively appear during early S phase and foci labelled early in one S phase colocalized with those labelled early in the next S phase. However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres. The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure. Replication initiated more frequently in the transcription unit than in the intergenic spacer. These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1-5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50-100 kb).

A dynamic stochastic model for DNA replication initiation in early embryos.

BACKGROUND: Eukaryotic cells seem unable to monitor replication completion during normal S phase, yet must ensure a reliable replication completion time. This is an acute problem in early Xenopus embryos since DNA replication origins are located and activated stochastically, leading to the random completion problem. DNA combing, kinetic modelling and other studies using Xenopus egg extracts have suggested that potential origins are much more abundant than actual initiation events and that the time-dependent rate of initiation, I(t), markedly increases through S phase to ensure the rapid completion of unreplicated gaps and a narrow distribution of completion times. However, the molecular mechanism that underlies this increase has remained obscure. METHODOLOGY/PRINCIPAL FINDINGS: Using both previous and novel DNA combing data we have confirmed that I(t) increases through S phase but have also established that it progressively decreases before the end of S phase. To explore plausible biochemical scenarios that might explain these features, we have performed comparisons between numerical simulations and DNA combing data. Several simple models were tested: i) recycling of a limiting replication fork component from completed replicons; ii) time-dependent increase in origin efficiency; iii) time-dependent increase in availability of an initially limiting factor, e.g. by nuclear import. None of these potential mechanisms could on its own account for the data. We propose a model that combines time-dependent changes in availability of a replication factor and a fork-density dependent affinity of this factor for potential origins. This novel model quantitatively and robustly accounted for the observed changes in initiation rate and fork density. CONCLUSIONS/SIGNIFICANCE: This work provides a refined temporal profile of replication initiation rates and a robust, dynamic model that quantitatively explains replication origin usage during early embryonic S phase. These results have significant implications for the organisation of replication origins in higher eukaryotes.

A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers.

Molecular combing of DNA is an extremely powerful DNA fiber-stretching technique that is often used in DNA replication and genome stability studies. Optimal DNA combing results mainly depend on the quality of the silanized surfaces onto which fibers are stretched. Here we describe an improved method of liquid-phase silanization using trimethoxy-octenylsilane/n-heptane as novel silane/solvent combination. Our simple method produces homogenously modified coverslips in a reproducible manner but does not require any sophisticated or expensive equipment in comparison to other known silanization protocols. However DNA fibers were combed onto these coverslips with very good high-density alignment and stayed irreversibly bound onto the surfaces after various denaturing treatments, as required for different immunofluorescent detection of DNA with incorporated modified nucleotides or FISH.

Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation.

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Delta/-) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system.

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G1 phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 +/- 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.

Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint.

A strict control of replication origin density and firing time is essential to chromosomal stability. Replication origins in early frog embryos are located at apparently random sequences, are spaced at close ( approximately 10-kb) intervals, and are activated in clusters that fire at different times throughout a very brief S phase. Using molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts, we show that the temporal order of origin firing can be modulated by the nucleocytoplasmic ratio and the checkpoint-abrogating agent caffeine in the absence of external challenge. Increasing the concentration of nuclei in the extract increases S phase length. Contrary to a previous interpretation, this does not result from a change in local origin spacing but from a spreading of the time over which distinct origin clusters fire and from a decrease in replication fork velocity. Caffeine addition or ATR inhibition with a specific neutralizing antibody increases origin firing early in S phase, suggesting that a checkpoint controls the time of origin firing during unperturbed S phase. Furthermore, fork progression is impaired when excess forks are assembled after caffeine treatment. We also show that caffeine allows more early origin firing with low levels of aphidicolin treatment but not higher levels. We propose that a caffeine-sensitive, ATR-dependent checkpoint adjusts the frequency of initiation to the supply of replication factors and optimizes fork density for safe and efficient chromosomal replication during normal S phase.

Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem.

Eukaryotic DNA replication initiates at multiple origins. In early fly and frog embryos, chromosomal replication is very rapid and initiates without sequence specificity. Despite this apparent randomness, the spacing of these numerous initiation sites must be sufficiently regular for the genome to be completely replicated on time. Studies in various eukaryotes have revealed that there is a strict temporal separation of origin "licensing" prior to S phase and origin activation during S phase. This may suggest that replicon size must be already established at the licensing stage. However, recent experiments suggest that a large excess of potential origins are assembled along chromatin during licensing. Thus, a regular replicon size may result from the selection of origins during S phase. We review single molecule analyses of origin activation and other experiments addressing this issue and their general significance for eukaryotic DNA replication.