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The full licensing of replication origins in late G1 is normally enforced by the licensing checkpoint. In this issue, Matson et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201902143) show that this checkpoint is inoperative in cells exiting from G0, resulting in incomplete origin licensing and consequent replicative stress., (© 2019 Blow.)

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.

In late mitosis and G 1 , 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 G 1 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 G 1 . 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., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

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.

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4-CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery-an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Mono-ubiquitination of Fancd2 is essential for repairing DNA interstrand cross-links (ICLs), but the underlying mechanisms are unclear. The Fan1 nuclease, also required for ICL repair, is recruited to ICLs by ubiquitinated (Ub) Fancd2. This could in principle explain how Ub-Fancd2 promotes ICL repair, but we show that recruitment of Fan1 by Ub-Fancd2 is dispensable for ICL repair. Instead, Fan1 recruitment--and activity--restrains DNA replication fork progression and prevents chromosome abnormalities from occurring when DNA replication forks stall, even in the absence of ICLs. Accordingly, Fan1 nuclease-defective knockin mice are cancer-prone. Moreover, we show that a Fan1 variant in high-risk pancreatic cancers abolishes recruitment by Ub-Fancd2 and causes genetic instability without affecting ICL repair. Therefore, Fan1 recruitment enables processing of stalled forks that is essential for genome stability and health., (Copyright © 2016, American Association for the Advancement of Science.)

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.

During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2-7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The ability of a eukaryotic cell to precisely and accurately replicate its DNA is crucial to maintain genome stability. Here we describe our current understanding of the process by which origins are licensed for DNA replication and review recent work suggesting that fork stalling has exerted a strong selective pressure on the positioning of licensed origins. In light of this, we discuss the complex and disparate phenotypes observed in mouse models and humans patients that arise due to defects in replication licensing proteins., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks.

Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin, still with high affinity (∼30 nm), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Only ∼10% of replication origins that are licensed by loading minichromosome maintenance 2-7 (MCM2-7) complexes are normally used, with the majority remaining dormant. If replication fork progression is inhibited, nearby dormant origins initiate to ensure that all of the chromosomal DNA is replicated. At the same time, DNA damage-response kinases are activated, which preferentially suppress the assembly of new replication factories. This diverts initiation events away from completely new areas of the genome toward regions experiencing replicative stress. Mice hypomorphic for MCM2-7, which activate fewer dormant origins in response to replication inhibition, are cancer-prone and are genetically unstable. The licensing checkpoint delays entry into S phase if an insufficient number of origins have been licensed. In contrast, humans with Meier-Gorlin syndrome have mutations in pre-RC proteins and show defects in cell proliferation that may be a consequence of chronic activation of the licensing checkpoint.

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. 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. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell-cycle-regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2-M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. By contrast, when Cdt1 is overexpressed or is stabilised by the neddylation inhibitor MLN4924, re-replication can occur throughout S phase.

DNA replication is an essential process in biology and its timing must be robust so that cells can divide properly. Random fluctuations in the formation of replication starting points, called origins, and the subsequent activation of proteins lead to variations in the replication time. We analyze these stochastic properties of DNA and derive the positions of origins corresponding to the minimum replication time. We show that under some conditions the minimization of replication time leads to the grouping of origins, and relate this to experimental data in a number of species showing origin grouping.

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pre-replication complexes (pre-RCs) are assembled onto DNA during late mitosis and G1 to license replication origins for use in S phase. In order to prevent re-replication of DNA, licensing must be completely shutdown prior to entry into S phase. While mechanisms preventing re-replication during S phase and mitosis have been elucidated, the means by which cells first prevent licensing during late G1 phase are poorly understood. We have employed a hybrid mammalian / Xenopus egg extract replication system to dissect activities that inhibit replication licensing at different stages of the cell cycle in Chinese Hamster Ovary (CHO) cells. We find that soluble extracts from mitotic cells inhibit licensing through a combination of geminin and Cdk activities, while extracts from S-phase cells inhibit licensing predominantly through geminin alone. Surprisingly however, geminin did not accumulate until after cells enter S phase. Unlike extracts from cells in early G1 phase, extracts from late G1 phase and early S phase cells contained an inhibitor of licensing that could not be accounted for by either geminin or Cdk. Moreover, inhibiting cyclin and geminin protein synthesis or inhibiting Cdk activity early in G1 phase did not prevent the appearance of inhibitory activity. These results suggest that a soluble inhibitor of replication licensing appears prior to entry into S phase that is distinct from either geminin or Cdk activity. Our hybrid system should permit the identification of this and other novel cell cycle regulatory activities.

Many replication origins that are licensed by loading MCM2-7 complexes in G1 are not normally used. Activation of these dormant origins during S phase provides a first line of defence for the genome if replication is inhibited. When replication forks fail, dormant origins are activated within regions of the genome currently engaged in replication. At the same time, DNA damage-response kinases activated by the stalled forks preferentially suppress the assembly of new replication factories, thereby ensuring that chromosomal regions experiencing replicative stress complete synthesis before new regions of the genome are replicated. Mice expressing reduced levels of MCM2-7 have fewer dormant origins, are cancer-prone and are genetically unstable, demonstrating the importance of dormant origins for preserving genome integrity. We review the function of dormant origins, the molecular mechanism of their regulation and their physiological implications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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.

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.

In this issue of Molecular Cell, Miotto and Struhl (2010) suggest that replication licensing, the loading of Mcm2-7 onto DNA, is promoted by HBO1 acetylating histone H4 at replication origins, providing a molecular view of how chromatin status influences origin usage., (Copyright 2010 Elsevier Inc. All rights reserved.)

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

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.

Correct regulation of the replication licensing system ensures that chromosomal DNA is precisely duplicated in each cell division cycle. Licensing proteins are inappropriately expressed at an early stage of tumorigenesis in a wide variety of cancers. Here we discuss evidence that misregulation of replication licensing is a consequence of oncogene-induced cell proliferation. This misregulation can cause either under- or over-replication of chromosomal DNA, and could explain the genetic instability commonly seen in cancer cells.

When DNA replication is slowed down, normally dormant replication origins are activated. Recent work demonstrates that cells adapt by changing the organization of chromatin loops and maintaining the new pattern of origin use in subsequent cell cycles.

Xenopus egg extract supports all the major cell-cycle transitions in vitro. We have used a proteomics approach to identify proteins whose abundance on chromatin changes during the course of an in vitro cell cycle. One of the proteins we identified was ELYS/MEL-28, which has recently been described as the earliest-acting factor known to be required for nuclear pore complex (NPC) assembly [1-4]. ELYS interacts with the Nup107-160 complex and is required for its association with chromatin. ELYS contains an AT-hook domain, which we show binds to chromatin with a high affinity. This domain can compete with full-length ELYS for chromatin association, thereby blocking NPC assembly. This provides evidence that ELYS interacts directly with chromatin and that this interaction is essential for NPC assembly and compartmentalization of chromosomal DNA within the cell. Furthermore, we detected a physical association on chromatin between ELYS and the Mcm2-7 replication-licensing proteins. ELYS chromatin loading, NPC assembly, and nuclear growth were delayed when Mcm2-7 was prevented from loading onto chromatin. Because nuclear assembly is required to shut down licensing prior to entry into S phase, our results suggest a mechanism by which these two early cell-cycle events are coordinated with one another.

Correct regulation of the replication licensing system ensures that no DNA is rereplicated in a single cell cycle. When the licensing protein Cdt1 is overexpressed in G2 phase of the cell cycle, replication origins are relicensed and the DNA is rereplicated. At the same time, checkpoint pathways are activated that block further cell cycle progression. We have studied the consequence of deregulating the licensing system by adding recombinant Cdt1 to Xenopus egg extracts. We show that Cdt1 induces checkpoint activation and the appearance of small fragments of double-stranded DNA. DNA fragmentation and strong checkpoint activation are dependent on uncontrolled rereplication and do not occur after a single coordinated round of rereplication. The DNA fragments are composed exclusively of rereplicated DNA. The unusual characteristics of these fragments suggest that they result from head-to-tail collision (rear ending) of replication forks chasing one another along the same DNA template.

Faithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs. We show that the formation of replication factories is a consequence of DNA replication itself. Our analyses of replication at specific DNA sequences support a long-standing hypothesis that sister replication forks generated from the same origin stay associated with each other within a replication factory while the entire replicon is replicated. This assay system allows replication to be studied at extremely high temporal resolution in individual cells, thereby opening a window into how replication dynamics vary from cell to cell.

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.

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.

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.

Background: Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase., Results: We have cloned a Xenopus homologue of Dbf4 (XDbf4), the sequence of which confirms the results of Furukhori et al. We have analysed the role of XDbf4 in DNA replication using cell-free extracts of Xenopus eggs. Our results indicate that XDbf4 is the regulatory subunit of XCdc7 required for DNA replication. We show that XDbf4 binds to chromatin during interphase, but unlike XCdc7, its chromatin association is independent of pre-RC formation, occurring in the absence of licensing, XCdc6 and XORC. Moreover, we show that the binding of XCdc7 to chromatin is dependent on the presence of XDbf4, whilst under certain circumstances XDbf4 can bind to chromatin in the absence of XCdc7. We provide evidence that the chromatin binding of XDbf4 that occurs in the absence of licensing depends on checkpoint activation., Conclusions: We have identified XDbf4 as a functional activator of XCdc7, and show that it is required to recruit XCdc7 to chromatin. Our results also suggest that XCdc7 and XDbf4 are differentially regulated, potentially responding to different cell cycle signals.

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.

To prevent re-replication of DNA, the licensing of replication origins is inhibited in S phase and G2. New work by Yamaguchi et al. (in this issue of Cell) shows that the small GTPase Ran can directly inhibit licensing inside nuclei once CDKs are active late in the cell cycle.