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Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the location of origin clusters on the genome has not been documented. Methods: We synchronised human U2OS by thymidine block and release followed by a brief block with L-mimosine to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped back onto the human genome. Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters were preferentially activated in sequence across a group, consistent with the ‘domino’ model of replication focus activation observed by microscopy. Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.

Cells face several challenges to completing genome duplication. One challenge is the irreversible stalling of converging replication forks (‘double fork stalls’). Cell types that cannot delay mitotic entry must also ensure that no replication origins are too far apart (the ‘random gap problem’). We show how these challenges can be met in early Xenopus embryos by the very abundant licensing of replication origins: one MCM2-7 double hexamer every ∼250 bp. Licensing does not change nucleosome spacing, consistent with MCM2-7 being assembled onto inter-nucleosomal linker DNA. We show that later embryonic development can occur successfully with a per-cell cycle failure rate of Xenopus early embryonic cells can ensure complete genome duplication without requiring unconventional replication mechanisms.

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. Mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, chromatin relaxation is controlled by SET8-dependent methylation of histone H4 on lysine 20. In the absence of either SET8 or H4K20 residue, substantial genome-wide chromatin decompaction occurs allowing excessive loading of the origin recognition complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase., SCOPUS: ar.j, info:eu-repo/semantics/published

During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place. The role of Mcm10 is particularly unclear in metazoans, where conflicting data has been presented. Here, we investigate the role and regulation of Mcm10 in Xenopus egg extracts. We show that Xenopus Mcm10 is recruited to chromatin late in the process of replication initiation and this requires prior action of DDKs and CDKs. We also provide evidence that Mcm10 is a CDK substrate but does not need to be phosphorylated in order to associate with chromatin. We show that in extracts depleted of more than 99% of Mcm10, the bulk of DNA replication still occurs, suggesting that Mcm10 is not required for the process of replication initiation. However, in extracts depleted of Mcm10, the replication fork elongation rate is reduced. Furthermore, the absence of Mcm10 or its phosphorylation by CDK results in instability of replisome proteins on DNA, which is particularly important under conditions of replication stress.

A key player in cancer prevention Obstacles that block DNA replication can lead to chromosomal abnormalities and ultimately cancer. Fanconi anemia, for example, is caused by defects in the repair of DNA interstrand cross-links. The Fan1 nuclease was originally identified as a protein essential for this cross-link repair. Lachaud et al . now show that it does much more. It is recruited to stalled replication forks by ubiquitinated Fancd2 enabling the processing of these structures, thereby preventing chromosomal abnormalities and acting more broadly in cancer prevention. Science , this issue p. 846

Here we discuss the important contributions that cell-free extracts have made to the study of complex biological processes. We provide a brief history of how cell-free extracts of frog eggs were developed to avoid many of the problems that can arise from the dilution and mixing of cellular components that typically occur when cell-free extracts are prepared. We briefly describe how Xenopus egg extracts have been fundamental to the study of many important cellular processes including DNA replication, cell cycle progression, nuclear protein import, nuclear assembly and chromosome organisation. We describe how, in particular, Xenopus egg extracts have made a major contributions to the study of DNA replication, by permitting the direct manipulation of proteins in a system that is extraordinarily faithful to the way that DNA replication occurs in the living embryo. Finally we consider how results obtained using Xenopus egg extracts are being translated to produce diagnostic reagents for cancer screening and diagnosis.

10 p.-2 tab., In order to maintain functional robustness and species integrity, organisms must ensure high fidelity of the genome duplication process. This is particularly true during early development, where cell division is often occurring both rapidly and coherently. By studying the extreme limits of suppressing DNA replication failure due to double fork stall errors, we uncover a fundamental constant that describes a trade-off between genome size and architectural complexity of the developing organism. This constant has the approximate value N_U ≈ 3×10^12 basepairs, and depends only on two highly conserved molecular properties of DNA biology. We show that our theory is successful in interpreting a diverse range of data across the Eukaryota., MAM, LA and TJN acknowledge prior support from the Scottish Universities Life Sciences Alliance. JJB acknowledges support from Cancer Research UK (grant C303/A14301) and the Wellcome Trust (grant WT096598MA). TJN acknowledges prior support from the National Institutes of Health (Physical Sciences in Oncology Centers, U54 CA143682).

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. The mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here, we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, appropriate chromatin relaxation is safeguarded by SET8-dependent methylation of histone H4 on lysine 20. Thus, in the absence of either SET8 or the H4K20 residue, substantial genome-wide chromatin decompaction occurs which allows excessive loading of the Origin Recognition Complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and the loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase.

During late mitosis and early G1, replication origins are licensed for replication by binding to double hexamers of MCM2-7. Here, we investigate how licensing and proliferative commitment are coupled in the small-intestinal epithelium. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5(+) stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem-cells is caused by an increased G1length, characterised by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost InApcmutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G1entry. We propose that the unlicensed G1of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.

Single-cell analyses in budding yeast reveal that neighboring replicons are assembled stochastically and stay associated to maintain stable replication factories., Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.

During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts-Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe-also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10(-8), which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms.

Summary Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3–Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2–Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites., Graphical Abstract, Highlights • The Ctf19 complex recruits Cdc7-Dbf4 kinase (DDK) to kinetochores (KTs) in telophase • DDK at KTs recruits Sld3–Sld7 to pericentromeric origins, advancing replication timing • DDK at KTs recruits Scc2, enhancing cohesin loading and cohesion at pericentromeres • The DDK functions in replication timing and cohesion are independent of each other

Hexameric complexes of the six related Mcm2-7 proteins form the core of the replicative helicase. Two other proteins, Mcm8 and Mcm9, with significant homology to Mcm2-7 were first shown to play distinct roles during DNA replication in Xenopus laevis egg extract. Recent work has revealed that Mcm8 and 9 form a complex that plays a role during homologous recombination in human, chicken and mouse cells. We have therefore re-examined the behavior of the Xenopus homologs of these proteins. We show that Mcm8 and Mcm9 form a dimeric complex in Xenopus egg extract. They both associate with chromatin at later stages of DNA replication, and this association is stimulated by DNA damage, suggesting that their function is analogous to the one described in higher eukaryotes. In contrast to previous reports, we do not find Mcm9 essential for loading of Mcm2-7 complex onto chromatin during origin licensing nor detect its interaction with Cdt1 origin licensing factor. Altogether, we conclude that the role Mcm8 and Mcm9 play in Xenopus egg extract is not different from recent findings in higher eukaryotes, consistent with an evolutionary conservation of their function.

To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.

The replication of DNA is initiated at particular sites on the genome called replication origins (ROs). Understanding the constraints that regulate the distribution of ROs across different organisms is fundamental for quantifying the degree of replication errors and their downstream consequences. Using a simple probabilistic model, we generate a set of predictions on the extreme sensitivity of error rates to the distribution of ROs, and how this distribution must therefore be tuned for genomes of vastly different sizes. As genome size changes from megabases to gigabases, we predict that regularity of RO spacing is lost, that large gaps between ROs dominate error rates but are heavily constrained by the mean stalling distance of replication forks, and that, for genomes spanning ∼100 megabases to ∼10 gigabases, errors become increasingly inevitable but their number remains very small (three or less). Our theory predicts that the number of errors becomes significantly higher for genome sizes greater than ∼10 gigabases. We test these predictions against datasets in yeast, Arabidopsis, Drosophila, and human, and also through direct experimentation on two different human cell lines. Agreement of theoretical predictions with experiment and datasets is found in all cases, resulting in a picture of great simplicity, whereby the density and positioning of ROs explain the replication error rates for the entire range of eukaryotes for which data are available. The theory highlights three domains of error rates: negligible (yeast), tolerable (metazoan), and high (some plants), with the human genome at the extreme end of the middle domain.

The DNA replication (or origin) licensing system ensures precise duplication of the genome in each cell cycle and is a powerful regulator of cell proliferation in metazoa. Studies in yeast, Drosophila melanogaster and Xenopus laevis have characterised the molecular machinery that constitutes the licensing system, but it remains to be determined how this important evolutionary conserved pathway is regulated in Homo sapiens. We have investigated regulation of the origin licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells. Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading the Mcm2-7 complex, which is required for unwinding the DNA helix, onto chromosomal origins. Geminin is a repressor of origin licensing that blocks Mcm2-7 loading onto origins. Our studies demonstrate that Cdc6, Cdt1 and Mcm2 play a central role in coordinating growth during the proliferation-differentiation switch in somatic self-renewing systems and that Cdc6 expression is rate-limiting for acquisition of replication competence in primary oocytes. In striking contrast, we show that proliferation control during male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated by the negative regulator Geminin with Cdt1 becoming rate-limiting in late prophase. Our data demonstrate a striking sexual dimorphism in the mechanisms repressing origin licensing and preventing untimely DNA synthesis during meiosis I, implicating a pivotal role for Geminin in maintaining integrity of the male germline genome.

The cyclin-dependent kinase inhibitor p21(WAF1/Cip1) is the prototype downstream effector of the tumor suppressor protein p53. Yet, evidence from human cancer and mice models, imply that p21(WAF1/Cip1), under certain conditions, can exercise oncogenic activity. The mechanism behind this behavior is still obscure. Within this context we unexpectedly noticed, predominantly in p53 mutant human cancers, that a subset of highly atypical cancerous cells expressing strongly p21(WAF1/Cip1) demonstrated also signs of proliferation. This finding suggests either tolerance to high p21(WAF1/Cip1) levels or that p21(WAF1/Cip1) per se guided a selective process that led to more aggressive off-springs. To address the latter scenario we employed p21(WAF1/Cip1)-inducible p53-null cellular models and monitored them over a prolonged time period, using high-throughput screening means. After an initial phase characterized by stalled growth, mainly due to senescence, a subpopulation of p21(WAF1/Cip1) cells emerged, demonstrating increased genomic instability, aggressiveness and chemo-resistance. At the mechanistic level unremitted p21(WAF1/Cip1) production “saturates” the CRL4(CDT2) and SCF(Skp2) ubiquitin ligase complexes reducing the turn-over of the replication licensing machinery. Deregulation of replication licensing triggered replication stress fuelling genomic instability. Conceptually, the above notion should be considered when anti-tumor strategies are designed, since p21(WAF1/Cip1) responds also to p53-independent signals, including various chemotherapeutic compounds.

Summary In late mitosis and G1, origins of DNA replication must be “licensed” for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2–7. A “licensing checkpoint” delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2–7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP., Graphical Abstract, Highlights • Arylquinolin-amines identified as small-molecule inhibitors of replication licensing • Inhibitor prevents tight ORC-DNA interaction required for MCM2–7 loading • ORC-DNA binding inhibited allosterically to mimic a lack of ATP, Gardner et al. describe the identification of a family of arylquinolin-amines as inhibitors of “replication licensing”, the loading of replication origins with double hexamers of MCM2–7. Arylquinolin-amines prevent the tight ORC-DNA interaction required for licensing, mimicking a lack of ATP.

Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We detail how these extracts can be used to study the key transitions of the eukaryotic cell cycle and describe conditions under which these transitions can be manipulated by addition of drugs that either retard or advance passage. In addition, we describe in detail essential techniques that provide a practical starting point for investigating the function of proteins involved in the operation of the eukaryotic cell cycle.

Cdt1 plays a key role in licensing DNA for replication. In the somatic cells of metazoans, both Cdt1 and its natural inhibitor geminin show reciprocal fluctuations in their protein levels owing to cell cycle-dependent proteolysis. Here, we show that the protein levels of Cdt1 and geminin are persistently high during the rapid cell cycles of the early Xenopus embryo. Immunoprecipitation of Cdt1 and geminin complexes, together with their cell cycle spatiotemporal dynamics, strongly supports the hypothesis that Cdt1 licensing activity is regulated by periodic interaction with geminin rather than its proteolysis. Overexpression of ectopic geminin slows down, but neither arrests early embryonic cell cycles nor affects endogenous geminin levels; apparent embryonic lethality is observed around 3-4 hours after mid-blastula transition. However, functional knockdown of geminin by ΔCdt1_193-447, which lacks licensing activity and degradation sequences, causes cell cycle arrest and DNA damage in affected cells. This contributes to subsequent developmental defects in treated embryos. Our results clearly show that rapidly proliferating early Xenopus embryonic cells are able to regulate replication licensing in the persistent presence of high levels of licensing proteins by relying on changing interactions between Cdt1 and geminin during the cell cycle, but not their degradation.

Summary Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication, CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors, including CDC-45 and GINS. During the elongation step, the CDC-45/GINS complex moves with the replication fork; however, it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48 UFD-1/NPL-4 -deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97.

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to license them for initiation in the upcoming S phase. After initiation, Mcm2-7 provide helicase activity to unwind DNA at the replication fork. Here we examine the structure of Mcm2-7 on chromatin in Xenopus egg extracts. We show that prior to replication initiation, Mcm2-7 is present at licensed replication origins in a complex with a molecular mass close to double that of the Mcm2-7 hexamer. This complex has approximately stoichiometric quantities of the 6 Mcm2-7 proteins and we conclude that it consists of a double heterohexamer. This provides a configuration potentially capable of initiating a pair of bidirectional replication forks in S phase. We also show that after initiation, Mcm2-7 associate with Cdc45 and GINS to form a relatively stable CMG (Cdc45-MCM-GINS) complex. The CMG proteins also associate less strongly with other replication proteins, consistent with the idea that a single CMG complex forms the core of the replisome.

At low levels of replication stress, Chk1 favors resolving problems at stalled replication forks over initiating origin firing in unreplicated areas of the genome., Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3–related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.

Cdk activity can differentially regulate the number of active replication factories, replication rates, and the rate of progression through the timing program during S phase., In the metazoan replication timing program, clusters of replication origins located in different subchromosomal domains fire at different times during S phase. We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei. Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it. Lowering cyclin-dependent kinase (Cdk) activity slowed both replication rate and progression through the timing program, whereas raising Cdk activity increased them. Surprisingly, modest alteration of Cdk activity changed the amount of DNA synthesized during different stages of the timing program. This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal. The ability of Cdks to differentially effect replication initiation, factory activation, and progression through the timing program provides new insights into the way that chromosomal DNA replication is organized during S phase.

Comment on: Origin licensing and p53 status regulate Cdk2 activity during G(1).Nevis KR, Cordeiro-Stone M, Cook JG

Replication origins are ‘licensed' for a single initiation event before entry into S phase; however, many licensed replication origins are not used, but instead remain dormant. The use of these dormant origins helps cells to survive replication stresses that block replication fork movement. Here, we present a computer model of the replication of a typical metazoan origin cluster in which origins are assigned a certain initiation probability per unit time and are then activated stochastically during S phase. The output of this model is in good agreement with experimental data and shows how inefficient dormant origins can be activated when replication forks are inhibited. The model also shows how dormant origins can allow replication to complete even if some forks stall irreversibly. This provides a simple explanation for how replication origin firing is regulated, which simultaneously provides protection against replicative stress while minimizing the cost of using large numbers of replication forks.

Monoubiquitination of proliferating cell nuclear antigen (PCNA) enables translesion synthesis (TLS) by specialized DNA polymerases to replicate past damaged DNA. We have studied PCNA modification and chromatin recruitment of TLS polymerases in Xenopus egg extracts and mammalian cells. We show that Xenopus PCNA becomes ubiquitinated and sumoylated after replication stress induced by UV or aphidicolin. Under these conditions the TLS polymerase eta was recruited to chromatin and also became monoubiquitinated. PTIP/Swift is an adaptor protein for the ATM/ATR kinases. Immunodepletion of PTIP/Swift from Xenopus extracts prevented efficient PCNA ubiquitination and polymerase eta recruitment to chromatin during replicative stress. In addition to PCNA ubiquitination, efficient polymerase eta recruitment to chromatin also required ATR kinase activity. We also show that PTIP depletion from mammalian cells by RNAi reduced PCNA ubiquitination in response to DNA damage, and also decreased the recruitment to chromatin of polymerase eta and the recombination protein Rad51. Our results suggest that PTIP/Swift is an important new regulator of DNA damage avoidance in metazoans.

Summary Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic. Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either replication licensing or S phase CDK activity. This revealed an unexpectedly broad system-wide effect on the chromatin proteome, indicating that the response to replication inhibition extends to many other functional modules in addition to the replication machinery. Several proteins that respond to replication inhibition (including nuclear pore proteins) coprecipitated with the Mcm2–7 licensing complex on chromatin, suggesting that Mcm2–7 play a central role in coordinating nuclear structure with DNA replication.

Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We detail how these extracts can be used to study the key transitions of the eukaryotic cell cycle and describe conditions under which these transitions can be manipulated by addition of drugs that either retard or advance passage. In addition, we describe in detail essential techniques that provide a practical starting point for investigating the function of proteins involved in the operation of the eukaryotic cell cycle.

Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis., Author Summary Meiosis is a special form of cell division needed for the formation of haploid gametes. During meiosis, DNA double-strand breaks are enzymatically induced and then repaired by the meiotic recombination pathway. Meiotic recombination is essential for genetic diversity and for accurate segregation of chromosomes during meiosis. Meiotic recombinational repair can either use the homolog or the sister chromatid as a template to generate different recombination intermediates. Recombination intermediates must be resolved or dissolved by specific endonucleases and the BLM helicase. Unresolved recombination intermediates lead to formation of chromatin bridges and perturb proper chromosome segregation. In this study we show that the concerted action of the BLM helicase HIM-6 and the evolutionarily conserved SMC-5/6 complex are important for the ordered processing of meiotic recombination intermediates, proper crossover (CO) formation and subsequent chromosome segregation in C. elegans. Notably, HIM-6 is required for the normal distribution of meiotic COs. We also propose that the interplay between SMC-5/6 and HIM-6 has a crucial role in the formation of a highly condensed and ordered chromosomal structure, which constrains BRC-1 dependent ectopic recombination.

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2–7 (Mcm2–7). The number of Mcm2–7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2–7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2–7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins ('DNA licensing') is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states.

During the cell-division cycle, chromosomal DNA must initially be precisely duplicated and then correctly segregated to daughter cells. The accuracy of these two events is maintained by two interlinked cycles: the replication licensing cycle, which ensures precise duplication of DNA, and the cohesion cycle, which ensures correct segregation. Here we provide a general overview of how these two systems are coordinated to maintain genetic stability during the cell cycle.

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2-7 proteins to form a pre-replicative complex. Mcm2-7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.

The mini-chromosome maintenance proteins Mcm2–7 are essential for DNA replication. They are loaded onto replication origins during G1 phase of the cell cycle to form a pre-replication complex (pre-RC) that licenses each origin for subsequent initiation. We have investigated the DNA elements that determine the dependence of yeast replication origins on Mcm2–7 activity, i.e. the sensitivity of an origin to mcm mutations. Using chimaeric constructs from mcm sensitive and mcm insensitive origins, we have identified two main elements affecting the requirement for Mcm2–7 function. First, transcription into an origin increases its dependence on Mcm2–7 function, revealing a conflict between pre-RC assembly and transcription. Second, sequence elements within the minimal origin influence its mcm sensitivity. Replication origins show similar differences in sensitivity to mutations in other pre-RC proteins (such as Origin Recognition Complex and Cdc6), but not to mutations in initiation and elongation factors, demonstrating that the mcm sensitivity of an origin is determined by its ability to establish a pre-RC. We propose that there is a hierarchy of replication origins with respect to the range of pre-RC protein concentrations under which they will function. This hierarchy is both ‘hard-wired’ by the minimal origin sequences and ‘soft-wired’ by local transcriptional context.

During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition.

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors--the origin recognition complex, Cdc6 and Cdt1--are required for origin licensing. We examine here how licensing is regulated in Xenopus egg extracts. We show that Cdt1 is downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase-promoting complex (APC/C), and inhibition by a protein called geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re-licensing and re-replication. The extent of re-replication is limited by checkpoint kinases that are activated as a consequence of re-replication itself. These results allow us to build a comprehensive model of how re-replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor.

In most eukaryotes, replication origins fire asynchronously throughout S-phase according to a precise timing programme. When replication fork progression is inhibited, an intra-S-phase checkpoint is activated that blocks further origin firing and stabilizes existing replication forks to prevent them undergoing irreversible collapse. We show that chromatin incubated in Xenopus egg extracts displays a replication-timing programme in which firing of new replication origins during S phase depends on the continued activity of S-phase-inducing cyclin-dependent kinases. We also show that low concentrations of the DNA-polymerase inhibitor aphidicolin, which only slightly slows replication-fork progression, strongly suppress further initiation events. This intra-S-phase checkpoint can be overcome by caffeine, an inhibitor of the ATM/ATR checkpoint kinases, or by neutralizing antibodies to ATR. However, depletion or inhibition of Chk1 did not abolish the checkpoint. We could detect no significant effect on fork stability when this intra-S-phase checkpoint was inhibited. Interestingly, although caffeine could prevent the checkpoint from being activated, it could not rescue replication if added after the timing programme would normally have been executed. This suggests that special mechanisms might be necessary to reverse the effects of the intra-S-phase checkpoint once it has acted on particular origins.

Before S phase, cells license replication origins for initiation by loading them with Mcm2-7 heterohexamers. This process is dependent on Cdc6, which is recruited to unlicensed origins. Using Xenopus egg extracts we show that although each origin can load many Mcm2-7 hexamers, the affinity of Cdc6 for each origins drops once it has been licensed by loading the first hexamers. This encourages the distribution of at least one Mcm2-7 hexamer to each origin, and thereby helps to ensure that all origins are licensed. Although Cdc6 is not essential for DNA replication once licensing is complete, Cdc6 regains a high affinity for origins once replication forks are initiated and Mcm2-7 has been displaced from the origin DNA. We show that the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition. These results show that Cdc6 plays multiple roles in ensuring precise chromosome duplication.