Geminin: A Major DNA Replication Safeguard in Higher Eukaryotes

Eukaryotes have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. These mechanisms prevent relicensing of origins of replication after initiation of DNA replication in S phase until the end of mitosis. Most of our knowledge of mechanisms controlling prereplication complex (pre-RC) formation are based on studies from yeast and Xenopus, while much less is known for mammalian cells. Here we discuss our recent data demonstrating that Geminin is required for preventing rereplication in human normal and cancer cells.     

Quality control in the initiation of eukaryotic DNA replication

Origins of DNA replication must be regulated to ensure that the entire genome is replicated precisely once in each cell cycle. In human cells, this requires that tens of thousands of replication origins are activated exactly once per cell cycle. Failure to do so can lead to cell death or genome rearrangements such as those associated with cancer. Systems ensuring efficient initiation of replication, while also providing a robust block to re-initiation, play a crucial role in genome stability. In this review, I will discuss some of the strategies used by cells to ensure once per cell cycle replication and provide a quantitative framework to evaluate the relative importance and efficiency of individual pathways involved in this regulation.     

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1α associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1α/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.     

Origins and complexes: the initiation of DNA replication

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.     

A p53-dependent checkpoint pathway prevents rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.     

ARS replication during the yeast S phase

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.     

Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control.

The mechanisms controlling duplication of the metazoan genome are only beginning to be understood. It is still unclear what organization of DNA sequences constitutes a chromosomal origin of DNA replication, and the regulation of origin activity during the cell cycle has not been fully revealed. We review recent results that indicate that chorion gene amplification in follicle cells of the Drosophila ovary is a model for investigating metazoan replication. Evaluation of cis sequence organization and function suggests that chorion loci share attributes with other replicons and provides insights into metazoan origin structure. Moreover, recent results indicate that chorion origins respond to S-phase control, but escape mechanisms that inhibit other origins from firing more than once in a cell cycle. Several identified genes that mediate amplification are critical for the cell cycle control of replication initiation. It is likely that further genetic screens for mutations that disrupt amplification will identify the cadre of proteins associated with origins and the regulatory pathways that control their activity. Furthermore, the recent development of methods to detect amplification in situ has uncovered new aspects of its developmental control. Examining this control will reveal links between developmental pathways and the cell cycle machinery. Visualization of amplifying chorion genes with high resolution also represents an opportunity to evaluate the influence of nuclear and chromosome structure on origin activity. The study of chorion amplification in Drosophila, therefore, provides great potential for the genetic and molecular dissection of metazoan replication.     

Replication origins in metazoan chromosomes: fact or fiction?

The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.     

DNA replication origins fire stochastically in fission yeast

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.     

A potential role for mini-chromosome maintenance (MCM) proteins in initiation at the dihydrofolate reductase replication origin.

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.     

The splicing factor SF2/ASF binds to ARS homologs in a human rDNA replication origin

The function of the relatively well-studied DNA replication origins in the yeast Saccharomyces cerevisiae is dependent upon interactions between origin replication complex (ORC) proteins and several defined origin sequence elements, including the 11 bp ARS consensus sequence (ACS). Although the ORC proteins, as well as numerous other protein components required for DNA replication initiation, are largely conserved between yeast and mammals, DNA sequences within mammalian replication origins are highly variable and sequences homologous to the yeast ACS elements are generally not present. We have previously identified several replication initiation sites within the nontranscribed spacer region of the human ribosomal RNA gene, and found that two highly utilized sites each contain a homologue of the yeast ACS embedded within a DNA unwinding element and a matrix attachment region. Here we examine protein binding within these initiation sites, and demonstrate that these ACS homologues specifically bind the alternate splicing factor SF2/ASF as well as GAPDH in vitro, and present evidence that the SF2/ASF interaction also occurs within the nuclei of intact cells. As the moderate upregulation of SF2/ASF has been linked to oncogenesis through the promotion of alternatively spliced forms of several regulatory proteins, our results suggest an additional mechanism by which SF2/ASF may influence the transformed cell phenotype.     

Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.     

The replicon revisited: an old model learns new tricks in metazoan chromosomes

The origins of DNA replication were proposed in the replicon model to be specified genetically by replicator elements that coordinate the initiation of DNA synthesis with gene expression and cell growth. Recent studies have identified DNA sequences in mammalian cells that fulfil the genetic criteria for replicators and are beginning to uncover the sequence requirements for the initiation of DNA replication. Mammalian replicators are com- posed of non-redundant modules that cooperate to direct initiation to specific chromosomal sites. Conversely, replicators do not show strong sequence similarity, and their ability to initiate replication depends on the chromosomal context and epigenetic factors, as well as their primary sequence. Here, we review the properties of metazoan replicators, and discuss the genetic and epigenetic factors that determine where and when DNA replication is initiated.     

Time to Be Versatile: Regulation of the Replication Timing Program in Budding Yeast

Eukaryotic replication origins are activated at different times during the S phase of the cell cycle, following a temporal program that is stably transmitted to daughter cells. Although the mechanisms that control initiation at the level of individual origins are now well understood, much less is known on how cells coordinate replication at hundreds of origins distributed on the chromosomes. In this review, we discuss recent advances shedding new light on how this complex process is regulated in the budding yeast Saccharomyces cerevisiae. The picture that emerges from these studies is that replication timing is regulated in cis by mechanisms modulating the chromatin structure and the subnuclear organization of origins. These mechanisms do not affect the licensing of replication origins but determine their ability to compete for limiting initiation factors, which are recycled from early to late origins throughout the length of the S phase.     

ATM and ATR Check in on Origins: A Dynamic Model for Origin Selection and Activation

Initiation of DNA replication occurs at origins of replication, traditionally defined by specific sequence elements. Sequence-dependent initiation of replication is the rule in prokaryotes and in the yeast Saccharomyces cereviseae. However, sequence-dependent initiation does not appear to be absolutely required in metazoan eukaryotes. Origin firing is instead likely dependent on stochastic initiation from chromatin-defined loci, despite the demonstration of some specific origins. Based on some recent observations in Xenopus laevis egg extracts and in mammalian cell culture, we propose that timing of origin firing is dependent on feedback from active replicons. This dynamic regulation of replication is mediated by sensing of ongoing replication by the DNA-damage checkpoint kinases ATM and ATR, which in turn downregulate neighboring and distal origins and replicons by inhibition of the S-phase kinases Cdk2 and Cdc7 and by inhibition of the replicative Mcm helicase. Origin selection, activation, and replicon progression are therefore constrained in both space and time via feedback from the cell cycle and ongoing replication.     

High‐resolution analysis of DNA synthesis start sites and nucleosome architecture at efficient mammalian replication origins

DNA replication origins are poorly characterized genomic regions that are essential to recruit and position the initiation complex to start DNA synthesis. Despite the lack of specific replicator sequences, initiation of replication does not occur at random sites in the mammalian genome. This has lead to the view that DNA accessibility could be a major determinant of mammalian origins. Here, we performed a high‐resolution analysis of nucleosome architecture and initiation sites along several origins of different genomic location and firing efficiencies. We found that mammalian origins are highly variable in nucleosome conformation and initiation patterns. Strikingly, initiation sites at efficient CpG island‐associated origins always occur at positions of high‐nucleosome occupancy. Origin recognition complex (ORC) binding sites, however, occur at adjacent but distinct positions marked by labile nucleosomes. We also found that initiation profiles mirror nucleosome architecture, both at endogenous origins and at a transgene in a heterologous system. Our studies provide a unique insight into the relationship between chromatin structure and initiation sites in the mammalian genome that has direct implications for how the replication programme can be accommodated to diverse epigenetic scenarios.