Search

Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin–MTBP is rate limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin–MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin–MTBP with TopBP1 which is a regulated crucial step in pre-initiation complex formation. These results suggest how DDK works together with CDKs to regulate Treslin–MTBP and plays a crucial in selecting which origins will undergo initiation.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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 late mitosis and G1, a complex of the essential initiation proteins Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other times licensing is inhibited by cyclin-dependent kinases (CDKs) and geminin, thus ensuring that origins fire only once per cell cycle. Here we show that, paradoxically, CDKs are also required to inactivate geminin and activate the licensing system. On exit from metaphase in Xenopus laevis egg extracts, CDK-dependent activation of the anaphase-promoting complex (APC/C) results in the transient polyubiquitination of geminin. This ubiquitination triggers geminin inactivation without requiring ubiquitin-dependent proteolysis, and is essential for replication origins to become licensed. This reveals an unexpected role for CDKs and ubiquitination in activating chromosomal DNA replication.

The proliferation of eukaryotic cells is a highly regulated process that depends on the precise duplication of chromosomal DNA in each cell cycle. Regulation of the replication licensing system, which promotes the assembly of complexes of proteins termed Mcm2-7 onto replication origins, is responsible for preventing re-replication of DNA in a single cell cycle. Recent work has shown how the licensing system is directly controlled by cyclin-dependent kinases (CDKs). Repression of origin licensing is emerging as a ubiquitous route by which the proliferative capacity of cells is lowered, and Mcm2-Mcm7 proteins show promise as diagnostic markers of early cancer stages. These results have prompted us to propose a functional distinction between the proliferative state and the non-proliferative state (including G0) depending on whether origins are licensed.

Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.

The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 inXenopusegg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replication origins early in S phase and is not limiting for progression through theXenopusreplication timing programme. We demonstrate that protein phosphatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously completed Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle. Over the last decade an enormous amount has been learned about how this is achieved. Much of the progress has come from genetic analysis in yeast. However, biochemical analysis of cell cycle events using extracts prepared from eggs of the South African clawed toad Xenopus laevis has also played an important role. Although there are differences in the detailed regulation of the yeast and frog cell cycles, the basic cell cycle machinery appears to have been conserved throughout evolution. The results obtained in these model organisms, therefore, seem likely to be generally applicable to the cell cycles of all eukaryotes. ### Xenopus eggs and egg extracts The fertilized Xenopus egg undergoes 12 synchronous rounds of cell division in ∼8 h. These cell divisions take place in the absence of growth, subdividing the large (∼1 mm diameter) single‐celled egg into ∼4000 smaller cells. Transcription does not occur during these early embryonic divisions, although translation of pre‐existing maternal mRNA continues. Most of the proteins required for cell cycle progression are pre‐formed in the egg, and the continuing translation of a single protein (cyclin B) can support passage through the whole cell cycle (Murray and Kirschner, 1989). The stockpile of cell cycle proteins present in the Xenopus egg became exploitable by biochemical means following the development of cell‐free extracts that support all the nuclear events of the early embryonic cell division cycle (Lohka and Masui, 1983). Gentle lysis associated with minimal dilution of the cytoplasm yields a ‘low speed supernatant’ that maintains all the cell cycle activities present in the intact egg. These ‘low speed supernatants’ support precise rounds of DNA replication on exogenously added DNA templates, and like the intact egg, only re‐replicate DNA after passage through …

Cell-free systems derived from unfertilized Xenopus eggs have been particularly informative in the study of the regulation and biochemistry of DNA replication. We have developed a Xenopus-based system to analyze proliferating cell nuclear antigen (PCNA)-specific effects on the functional properties of egg extracts. To do this, we have coupled peptides derived from p21 (Waf1/Cip1) to beads and used these to deplete PCNA from Xenopus egg extracts. The effect on various aspects of DNA replication can be analyzed after the readdition of PCNA and other purified proteins. Using this system, we have shown that replication of single-stranded M13 DNA is entirely dependent upon PCNA. By adding exogenous T7 DNA polymerase to PCNA-depleted extracts, we have uncoupled processive DNA replication from PCNA activity and so created an experimental system to analyze the dependence of postreplicative processes on PCNA function. We have shown that successful chromatin assembly is specifically dependent on PCNA. However, systems for analyzing the far more complex mechanisms required for the replication of nuclear double-stranded DNA have proved so far to be refractory to specific PCNA depletion.

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., Genes & Development, 27 (23), ISSN:0890-9369, ISSN:1549-5477

The initiation of DNA replication forks occurs at replication origins distributed along chromosomes and must be strictly controlled to ensure that the DNA is replicated once and once only in each cell cycle (for review, see Diffley 1996; Stillman 1996; Donaldson and Blow 1999). The process of building an active replication origin can be divided into two phases. The first phase, occurring in late mitosis and early G1, comprises the sequential assembly onto replication origins of the origin recognition complex (ORC), the Cdc6 protein, and the RLF–M complex of Mcm2–7 proteins (also known as MCM/P1 proteins), which results in them becoming “licensed” for DNA replication in the subsequent S phase. The second phase involves the action of the Cdc7 and cyclin-dependent kinases (CDKs) on each origin to load the Cdc45 protein and to induce the initiation of a pair of replication forks. It is of considerable current interest to understand in more detail the sequence of events leading to the initiation of replication. In this paper we have concentrated on defining the precise stage in the process when the Cdc7 protein acts, using the biochemically tractable Xenopus cell-free DNA replication system. Replication origins appear to be defined by binding the ORC (Bell and Stillman 1992). In yeast, ORC is bound to origins throughout the cell cycle (Diffley and Cocker 1992; Diffley et al. 1994), whereas in higher eukaryotes, ORC is probably displaced from the DNA during mitosis (Coleman et al. 1996; Romanowski et al. 1996; Rowles et al. 1999). During late mitosis and early G1, Cdc6 is then assembled onto ORC-containing DNA (Coleman et al. 1996). Chromatin containing ORC and Cdc6 can then be licensed by loading the RLF-M complex of MCM/P1 proteins, a reaction also requiring the RLF-B component of the replication licensing system (Chong et al. 1995; Kubota et al. 1995, 1997; Thommes et al. 1997; Tada et al. 1999; Prokhorova and Blow 2000). The complex of ORC, Cdc6, and MCM/P1 proteins appears to be responsible for the footprint of the prereplicative complex (pre-RC) observed in Saccharomyces cerevisiae on replication origins in late mitosis and early G1 (Diffley et al. 1994). Once licensing has occurred, ORC and Cdc6 become more loosely bound to DNA and have fulfilled their essential function in DNA replication (Hua and Newport 1998; Rowles et al. 1999). For a licensed origin to initiate replication, two S phase-promoting protein kinases are then required: an S phase-promoting CDK and the Cdc7/Dbf4 protein kinase. Cdc7 is a serine threonine kinase conserved from yeast to humans that is required for the initiation of DNA replication (Hollingsworth et al. 1992; Jackson et al. 1993; Masai et al. 1995; Jiang and Hunter 1997; Sato et al. 1997; Hess et al. 1998). Although Cdc7 protein levels are approximately constant throughout the cell cycle, Cdc7 kinase activity peaks at the G1/S transition (Jackson et al. 1993; Yoon et al. 1993). This regulation is achieved in part by association with a regulatory subunit termed Dbf4 (Brown and Kelly 1999; Cheng et al. 1999; Jiang et al. 1999; Oshiro et al. 1999; Takeda et al. 1999). Instead of acting as a general initiator of S phase, Cdc7 is probably required to promote initiation at individual origins because, as well as being required for progression into early S phase, it is also required late in S phase to promote initiation at late-firing origins (Bousset and Diffley 1998; Donaldson et al. 1998a). Several lines of evidence suggest that the MCM/P1 proteins are the physiological substrate of Cdc7/Dbf4. In yeast, a mutant allele of Mcm5 (mcm5–bob1) can suppress a complete loss of Cdc7 or Dbf4 (Hardy et al. 1997). Conversely, a mutant of Dbf4 (dbf4-6) was isolated as an allele-specific suppressor of Mcm2 (Lei et al. 1997). Cdc7 and Dbf4 interact physically with MCM/P1 proteins (Lei et al. 1997; Roberts et al. 1999), and a number of reports have identified MCM/P1 proteins as excellent substrates of Cdc7/Dbf4 kinase in vitro (Lei et al. 1997; Sato et al. 1997). Moreover, certain phosphorylations of the MCM/P1 proteins (most notably of Mcm2) in vivo depend on Cdc7 (Lei et al. 1997; Jiang et al. 1999). The ability of Cdc7 to phosphorylate the MCM/P1 proteins may be aided by its recruitment to replication origins because, in yeast, both Dbf4 and Cdc7 have been shown to interact with chromatin (Pasero et al. 1999; Weinreich and Stillman 1999), the association being dependent on ORC but not Cdc6 (Pasero et al. 1999). This interaction may be mediated by Dbf4, because a one-hybrid screen identified a domain of Dbf4 distinct from the Cdc7 interaction domain that recruited Dbf4 to replication origins (Dowell et al. 1994). Cdc7/Dbf4 could be a target of checkpoint kinases, because when yeast cells were treated with hydroxyurea to block progression through S phase, Dbf4 dissociated from the chromatin (Pasero et al. 1999). This treatment also induced the appearance of a phosphorylated form of Dbf4, which was dependent on the checkpoint kinase Rad53 (Brown and Kelly 1999; Cheng et al. 1999; Takeda et al. 1999; Weinreich and Stillman 1999). CDKs are also required for the initiation of licensed replication origins. CDK activity leads to the assembly of the essential initiation protein Cdc45 onto origins, creating a “preinitiation complex” (Zou and Stillman 1998). Cdc45 interacts genetically with different components of the pre-RC including MCM/P1 proteins and ORC (Hopwood and Dalton 1996; Owens et al. 1997; Zou et al. 1997), and the association of Cdc45 with chromatin requires Cdc6 and Mcm2 (Zou and Stillman 1998). However, the binding of yeast Cdc45 to chromatin is independent of Cdc7 function, suggesting that CDKs and Cdc7 may act on parallel pathways to initiate replication (Zou and Stillman 1998). CDKs play an important role in executing the temporal program of origin activation during the course of S phase (Donaldson et al. 1998b). Like Cdc7, CDKs are probably required to promote initiation at individual origins, because the CDK-dependent loading of Cdc45 onto late-firing origins only occurs late in S phase (Aparicio et al. 1999). In the present report, we have used cell-free extracts of Xenopus eggs to determine the precise stage in the process of origin activation at which Xenopus (X) Cdc7 acts. We show that XCdc7 binds to chromatin during G1 and S phase and that both the chromatin binding and the essential DNA replication function of XCdc7 are dependent on licensing but do not require the presence of XORC or XCdc6. We show that XCdc7 is required for the subsequent loading of XCdc45 onto chromatin by CDKs. Finally we show that XCdc7-dependent phosphorylation of XMcm2 and the essential function of XCdc7 can be performed in the absence of CDK activity. These results provide a simple model for the function of Cdc7 in the Xenopus system that appears to differ from that occurring in yeast.

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

The origin recognition complex (ORC) binds to origins of replication in budding yeast. We have cloned a Xenopus homolog of the largest ORC polypeptide (XORC1). Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication. We have purified Xenopus ORC, consisting of a protein complex similar to yeast ORC. In Xenopus egg extracts, ORC associates with chromatin throughout G1 and S phases. RLF-M, a component of the replication licensing system, also associates with chromatin early in the cell cycle but dissociates during S phase. We show that the assembly of RLF-M onto chromatin is dependent on the presence of chromatin-bound ORC, leading to sequential assembly of initiation proteins onto replication origins during the cell cycle.

The DNA replication licensing system ensures that eukaryotic chromosomes replicate precisely once per cell cycle. A central component of the licensing system, RLF-M, has recently been shown to consist of a complex of Mcm/P1 proteins. This result allows us to integrate data about the MCM/P1 family obtained in different eukaryotes, ranging from yeast to man, into a general picture of the way that chromosome replication is controlled.

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.

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.

Live imaging in Caenorhabditis elegans provides an unprecedented view of the chromatin-binding behavior and subcellular localization of origin replication licensing proteins during early embryogenesis., 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.

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. In contrast, when Cdt1 is overexpressed or is stabilised by the Neddylation inhibitor MLN4924, re-replication can occur throughout S phase.

Background: DNA that is damaged by ultraviolet (UV) light is repaired predominantly by nucleotide excision–repair, a process requiring the DNA polymerase auxiliary factor PCNA. UV-irradiation also induces the production of Cip1 protein via activation of p53. Cip1 is an inhibitor of the cyclin-dependent kinases, which are required for the cell cycle to proceed through the G1/S-phase transition and initiate DNA replication. Inhibition by Cip1 probably causes the block to initiation of DNA replication that is seen in irradiated cells. Cip1 also directly inhibits the function of PCNA during DNA synthesis. As nucleotide excision–repair requires PCNA, the physiological relevance of PCNA inhibition by Cip1 is currently unclear. Results We show that nucleotide excision–repair of UV-damaged DNA occurs in extracts of Xenopus eggs, and that this reaction is PCNA-dependent. The repair reaction is not inhibited by Cip1, even when the level of PCNA is reduced 100-fold so that it becomes limiting for DNA repair. By contrast, Cip1 strongly suppresses the function of PCNA in replicative DNA synthesis under these conditions. Conclusion Cip1 can potentially inhibit DNA replication in Xenopus egg extracts by inhibiting the cyclin-dependent kinase function required for the initiation of replication forks, and also by inhibiting PCNA function. The inhibition of PCNA is selective for its function in DNA replication, however, as Cip1 does not affect PCNA function in nucleotide excision–repair. The induction of Cip1 in response to DNA damage, therefore, allows repair to continue in the genome under conditions in which replication is severely inhibited.

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.

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.

During S-phase of the cell cycle, chromosomal DNA is replicated according to a complex replication timing program, with megabase-sized domains replicating at different times. DNA fiber analysis reveals that clusters of adjacent replication origins fire near-synchronously. Analysis of replicating cells by light microscopy shows that DNA synthesis occurs in discrete foci or factories. The relationship between timing domains, origin clusters and replication foci is currently unclear. Recent work, using a hybrid Xenopus/hamster replication system, has shown that when CDK levels are manipulated during S-phase the activation of replication factories can be uncoupled from progression through the replication timing program. Here, we use data from this hybrid system to investigate potential relationships between timing domains, origin clusters and replication foci. We suggest that each timing domain typically comprises several replicon clusters, which are usually processed sequentially by replication factories. We discuss how replication might be regulated at different levels to create this complex organization and the potential involvement of CDKs in this process.

Proceedings of the National Academy of Sciences of the United States of America 106, 19807-19812 (2009). doi:10.1073/pnas.0905281106, Published by Academy, Washington, DC

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.

Summary 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.

Replication origins are 'licensed' for a single initiation event by loading Mcm2-7 complexes during late mitosis and G1. Licensing is blocked at other cell cycle stages by the activity of cyclin-dependent kinases and a small protein called geminin. Here, we describe the effects of over-expressing a non-degradable form of geminin in various cell lines. Geminin expression reduced the quantity of Mcm2 bound to chromatin and blocked cell proliferation. U2OS (p53+/Rb+) cells showed an early S phase arrest with high cyclin E and undetectable cyclin A levels, consistent with the activation of an intra-S checkpoint. Saos2 (p53-/Rb-) cells showed an accumulation of cells in late S and G2/M with approximately normal levels of cyclin A, consistent with loss of this intra-S phase checkpoint. Geminin also induced apoptosis in both these cell lines. In contrast, IMR90 primary fibroblasts over-expressing geminin arrested in G1 with reduced cyclin E levels and no detectable apoptosis. A 'licensing checkpoint' may therefore act in primary cells to prevent passage into S phase in the absence of sufficient origin licensing. These results suggest that inhibition of the licensing system may cause cancer-specific cell killing and therefore represent a novel anti-cancer target.

The initiation of DNA replication is restrained by Cip/Kip proteins that inhibit Cdk2. Degradation of Xenopus Xic1, a Kip1 orthologue, is dependent on its recruitment to replication origins. This ensures that activation of Cdk2 and (subsequent initiation of replication) is co-ordinately regulated at, and localised to, replication origins.

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.

Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

Publisher Summary This chapter discusses the characterization of the xenopus replication licensing system. In eukaryotes the entire genome is replicated precisely once during each S phase of the cell cycle. Cell-free extracts of Xenopus eggs support chromosomal DNA replication in vitro, apparently under the same cell cycle controls that exist in vivo. When DNA is added to the cell-free system it is assembled into chromatin, and then into structures resembling normal interphase nuclei, processes that is necessary before the initiation of DNA replication can occur. The precise duplication of chromosomal DNA in the Xenopus cell-free system requires many thousands of replication origins to each fire once and only once. To achieve this, two distinct signals are required for initiation to occur at any given replication origin.

Summary In order for cells to divide, they must replicate their chromosomes precisely once. This replication is performed by a number of proteins that act together to form a replication fork. The major control over DNA replication occurs at replication origins, the sites where active replication forks are initiated. In the yeast S. cerevisiae, origins have been identified as ARSs which possess a defined DNA sequence. These ARSs are recognized by specific proteins including ORC and ABF1. The elements which define origins in higher eukaryotes have not yet been identified. The temporal sequence in which origins fire is dependent on both the chromosomal location of the origin and the transcriptional activity of the dividing cell, with euchromatin and actively-expressed genes tending to replicate early in S-phase. DNA replication is an all-or-nothing event and occurs only after commitment to cell division. Cell fusion studies imply that S-phase inducers must be supplied to a nucleus as a signal to start replication. In addition, cells which have replicated their DNA but not yet undergone mitosis, will not perform further replication, even in the presence of these S-phase inducers. This is currently best explained by the action of a licensing factor, an origin-binding protein which cannot cross the nuclear envelope and so requires a transient nuclear permeabilization in order to make the DNA replication competent. Cyclin-dependent kinases provide good candidates for the S-phase inducers, as cell-cycle regulated activities which can potentially act upon these origin complexes and actually induce initiation to take place. Precise identification of the proteins involved is still to be made.

DNA Replication Licensing Factor (RLF) is an essential activity required to restrict the duplication of genomic DNA to precisely once per cell cycle. Recent fractionation of RLF activity from Xenopus egg extracts has resulted in the identification of two essential components, RLF-B and RLF-M. RLF-M has been purified to homogeneity and has been shown to consist of a complex of proteins in the MCM/P1 family. RLF-B is still unidentified, but possible candidates for this activity have been identified in yeast. Elucidation of the RLF mechanism will provide important insights into the way that chromosome replication is controlled.