Bidirectional Replication Forks Research Articles

DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation.

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Our data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.

Where and when to start: Regulating DNA replication origin activity in eukaryotic genomes

ABSTRACT In eukaryotic genomes, hundreds to thousands of potential start sites of DNA replication named origins are dispersed across each of the linear chromosomes. During S-phase, only a subset of origins is selected in a stochastic manner to assemble bidirectional replication forks and initiate DNA synthesis. Despite substantial progress in our understanding of this complex process, a comprehensive ‘identity code’ that defines origins based on specific nucleotide sequences, DNA structural features, the local chromatin environment, or 3D genome architecture is still missing. In this article, we review the genetic and epigenetic features of replication origins in yeast and metazoan chromosomes and highlight recent insights into how this flexibility in origin usage contributes to nuclear organization, cell growth, differentiation, and genome stability.

H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins.

The transmission and maintenance of genetic information in eukaryotic cells relies on the faithful duplication of the entire genome. In each round of division, excessive replication origins are licensed, with only a fraction activated to give rise to bi-directional replication forks in the context of chromatin. However, it remains elusive how eukaryotic replication origins are selectively activated. Here we demonstrate that O-GlcNAc transferase (OGT) enhances replication initiation by catalyzing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing reduced phosphorylation of the replicative helicase mini-chromosome maintenance (MCM) complex and compromised DNA unwinding. Our short nascent-strand sequencing results further confirm the importance of H4S47 O-GlcNAcylation in origin activation. We propose that H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the control of replication efficiency by chromatin environment.

Dimerization of Firing Factors for Replication Origin Activation in Eukaryotes: A Crucial Process for Simultaneous Assembly of Bidirectional Replication Forks?

Simple SummaryChromosomal DNA must be faithfully duplicated and segregated into two daughter cells when cells divide. DNA synthesis initiates from specific regions known as the origins of replication. When it starts, a pair of the replication fork is established, and each replication fork moves away from replication origins. In each replication fork, replicative helicase unwinds DNA from a double to a single strand. This implies that two sets of active helicase are generated from each replication origin. To make this possible, two sets of replicative helicases are loaded onto replication origins as inactive dimers first. When S-phase specific cyclin-dependent kinases, S-CDKs, are activated, the inactive helicase is converted into the active helicase with the aid of other factors called firing factors. Although two sets of firing factors seem to be required to activate two sets of helicase, it is largely unknown whether two sets of firing factors function simultaneously to establish bidirectional replication forks in a coordinated way. We introduce our current understanding of firing factor dimerization and discuss its potential contribution to bidirectional replication fork formation in this review.Controlling the activity of the heterohexameric Mcm2–7 replicative helicase is crucial for regulation of replication origin activity in eukaryotes. Because bidirectional replication forks are generated from every replication origin, when origins are licensed for replication in the first step of DNA replication, two inactive Mcm2–7 heterohexiameric complexes are loaded around double stranded DNA as a head-to-head double hexamer. The helicases are subsequently activated via a ‘firing’ reaction, in which the Mcm2–7 double hexamer is converted into two active helicase units, the CMG complex, by firing factors. Dimerization of firing factors may contribute to this process by allowing simultaneous activation of two sets of helicases and thus efficient assembly of bidirectional replication forks. An example of this is dimerization of the firing factor Sld3/Treslin/Ticrr via its binding partner, Sld7/MTBP. In organisms in which no Sld7 ortholog has been identified, such as the fission yeast Schizosaccharomyces pombe, Sld3 itself has a dimerization domain, and it has been suggested that this self-interaction is crucial for the firing reaction in this organism. Dimerization induces a conformational change in Sdl3 that appears to be critical for the firing reaction. Moreover, Mcm10 also seems to be regulated by self-interaction in yeasts. Although it is not yet clear to what extent dimerization of firing factors contributes to the firing reaction in eukaryotes, we discuss the possible roles of firing factor dimerization in simultaneous helicase activation.

Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes.

Human papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCE Human papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.

Bayesian inference of origin firing time distributions, origin interference and licencing probabilities from Next Generation Sequencing data.

DNA replication is a stochastic process with replication forks emanating from multiple replication origins. The origins must be licenced in G1, and the replisome activated at licenced origins in order to generate bi-directional replication forks in S-phase. Differential firing times lead to origin interference, where a replication fork from an origin can replicate through and inactivate neighbouring origins (origin obscuring). We developed a Bayesian algorithm to characterize origin firing statistics from Okazaki fragment (OF) sequencing data. Our algorithm infers the distributions of firing times and the licencing probabilities for three consecutive origins. We demonstrate that our algorithm can distinguish partial origin licencing and origin obscuring in OF sequencing data from Saccharomyces cerevisiae and human cell types. We used our method to analyse the decreased origin efficiency under loss of Rat1 activity in S. cerevisiae, demonstrating that both reduced licencing and increased obscuring contribute. Moreover, we show that robust analysis is possible using only local data (across three neighbouring origins), and analysis of the whole chromosome is not required. Our algorithm utilizes an approximate likelihood and a reversible jump sampling technique, a methodology that can be extended to analysis of other mechanistic processes measurable through Next Generation Sequencing data.

Destabilization of the MiniChromosome Maintenance (MCM) complex modulates the cellular response to DNA double strand breaks

ABSTRACTDNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is replicated only once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both the initiation and elongation of DNA replication. Although only two DNA helicase activities are necessary to establish a bidirectional replication fork from each replication origin, a large excess of MCM complexes is amassed and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most are displaced from the DNA during the S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of a reduction in MCM complex. The alteration of MCM proteins induced a change in the activation of key factors of the DDR in response to Etoposide treatment. Etoposide-induced DNA damage affected the phosphorylation of γ-H2AX, CHK1 and CHK2 without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we identified a decrease in both HR and NHEJ associated with a decrease in MCM complex.

Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier.

Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative “short tract” gene conversion (STGC) induced by a chromosomal Tus/Ter RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/Ter-induced STGC proceeds via a two-ended DSB intermediate. If so, Tus/Ter-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/Ter-induced HR is unaffected by deletion of either of two C-NHEJ genes, Xrcc4 or Ku70. These results show that Tus/Ter-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase θ, competes with Tus/Ter-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/Ter RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/Ter was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/Ter was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/Ter RFB are not conventional DSBs. We propose that Rad51 acts as an “early responder” at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery.

The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism.

The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells

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

Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes

Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polα, PCNA, topoisomerase, Cdc45, polδ, and polɛ are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.

Mechanisms of Chromosomal DNA Replication Initiation and Stabilization of Replication Forks in Eukaryotes

In eukaryotes, DNA replication initiates at multiple and specific sites on chromosomal DNA. When cells are in late M and G1 phases, Cdc6/Cdc18, Cdt1 and MCM are loaded to ORC binding sites on DNA replication origins to form pre-RC (pre-replication complex). Pre-RC is activated at the transition of G1 to S phase. Subsequently, those proteins that act at replication forks bind to DNA origins to form two bidirectional replication forks for initiating DNA synthesis. On chromosome, the moving replication forks often encounter replication fork barriers (DNA secondary structure, some protein binding sites, damaged bases) and stall. The stalled forks require checkpoint control to maintain fork stability; otherwise, stalled forks will collapse and genomic integrity is compromised. This article gives a brief review about current understanding of the mechanism of replication initiation and replication fork stabilization.

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex (pre-IC). This complex formation facilitates the conversion of Mcm2-7 in the pre-RC to an active DNA helicase, the Cdc45-Mcm-GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.

MCM2-7 Form Double Hexamers at Licensed Origins in Xenopus Egg Extract

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.

A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.

Escherichia coli DnaA Protein Loads a Single DnaB Helicase at a DnaA Box Hairpin

The molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB. At oriC, two and only two helicase molecules are loaded, one for each replication fork. DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase. Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind. In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused. We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure.

The extrusion-capture model for chromosome partitioning in bacteria.

Successful cellular reproduction requires accurate duplication and partitioning (segregation) of the genome. Failure to correctly partition the sister genomes results in aneuploidy. The consequences of these errors range from loss of normal cellular function (e.g., the loss of normal growth controls in tumor cells) to cell death. In prokaryotes with a single chromosome, partitioning failures are fatal for at least one of the two daughter cells; a so-called anucleate cell forms when one daughter receives no chromosome and the other daughter receives two chromosomes. Several findings in recent years have fundamentally altered our view of chromosome partitioning in prokaryotes (for review, see Gerdes et al. 2000; Gordon and Wright 2000; Hiraga 2000; Moller-Jensen et al. 2000; Donachie 2001; Sawitzke and Austin 2001). The flurry of new observations was ignited by adaptation of cell biological techniques used in eukaryotes (immunofluorescence, GFP, and fluorescent in situ hybridization) for use in prokaryotes (Harry et al. 1995; Pogliano et al. 1995; Webb et al. 1995; Niki and Hiraga 1998). This review focuses on chromosome partitioning in bacteria that have a single circular chromosome, specifically, the gram-positive organism, Bacillus subtilis, and the gramnegative organisms, Escherichia coli and Caulobacter crescentus. In these bacteria, DNA replication initiates once per cell division cycle from a specific chromosomal locus, oriC, and proceeds bidirectionally to terminate in a defined region opposite the origin, terC (Fig. 1). The basic components of the DNA replication machinery are highly conserved in bacteria (Kornberg and Baker 1992); in fact, these basic components are functionally conserved from bacteria to mammals (Baker and Bell 1998). Fundamental differences exist between chromosome partitioning in eukaryotes and prokaryotes. In eukaryotes, chromosomes are duplicated in S phase, and sister chromosomes remain together during G2. Chromosome partitioning occurs during M phase, when sister chromosomes are lined up on a metaphase plate, separated from each other, and finally segregated in opposite directions by the combined action of the microtubular spindle and mitotic motors. In contrast to the temporal separation of chromosome replication and partitioning in eukaryotes, regions of the bacterial chromosome, starting with the origin, appear to be partitioned soon after duplication, whereas the remainder of the chromosome awaits replication (Glaser et al. 1997; Gordon et al. 1997; Lewis and Errington 1997; Lin et al. 1997; Mohl and Gober 1997; Webb et al. 1997, 1998; Niki and Hiraga 1998; Sharpe and Errington 1998; Teleman et al. 1998; Jensen and Shapiro 1999; Niki et al. 2000). Thus, in bacteria, DNA replication, chromosome refolding, and chromosome partitioning are concurrent. Given these differences, it is not surprising that there is no evidence that bacteria contain eukaryotic-like mitotic spindles or mitotic motors. As discussed below, it appears that bacteria with circular chromosomes probably power chromosome partitioning differently from eukaryotes. This may be possible because of the much smaller distances that chromosomes move in bacteria. Many bacteria, including B. subtilis and E. coli, are capable of dividing in one-half to one-third of the time it takes to duplicate the genome. To accomplish this, new rounds of DNA replication are initiated before a previous round is completed, giving the replication cycle a head start on the division cycle. This results in cells with multiple bidirectional DNA replication forks (so-called multifork replication), multiple copies of oriC (2, 4, or 8), but only a single, unduplicated terminus region (Fig. 2). Soon after duplication, sister origins are each partitioned in opposite directions. Thus, during multifork replication, bacteria contain positional information regarding not only the current medial division site, but also the future division planes (the cell quarters and cell eighths). Other bacteria, for exampleC. crescentus, are not known to be capable of multifork replication and do not have more than two copies of the origin per cell. Recent studies have revealed common mechanisms involved in chromosome partitioning, along with differences in some of the details between organisms. This review summarizes the following recent findings that have contributed new insights into the mechanism by which bacteria accomplish accurate chromosome partitioning. First, the bacterial chromosome has a defined orientation within cells, and individual regions move 3Corresponding author. E-MAIL adg@mit.edu; FAX (617) 253-2643. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.913301.

ORC binding, gene amplification, and the nature of metazoan replication origins.

Genomic DNA replication is a critical process shaped by strong evolutionary pressures. Rapid replication can give a viral or cellular genome an immediate advantage over its competitors as long as requisitely high accuracy is not sacrificed. Prokaryotes employ an extremely fast, accurate DNA replication mechanism utilizing sophisticated multiprotein machines (Kornberg and Baker 1992; Baker and Bell 1998). Genome duplication is controlled by the assembly and activation of bidirectional replication forks at specific replication origins, under the control of initiator proteins that bind origins sequence specifically. For example, Escherichia coli oriC binds multiple copies of the initiator protein, dnaA, at repeated motifs. In an ATP-dependent manner dnaA then locally unwinds the DNA, facilitates helicase loading, and organizes the assembly of polymerases, primases, and other components of the replication fork. Special proteins are required to assemble and maintain this unwieldy machinery. Thus, loading the ring-shaped six-protein dnaB helicase around the DNA requires dnaC, whereas proteins such as tau probably act as scaffolds, organizing replication fork components (including polymerases). Despite the greater complexity of eukaryotic chromosomes, work carried out in Saccharomyces cerevisiae indicates that its genome replicates via a strongly similar mechanism (Leatherwood 1998). Each yeast chromosome contains multiple discrete replication origins where prereplication complexes are assembled during late M and G1 phases. Fully formed, bidirectional replication forks are then activated during S phase. Replication origins can be readily identified as autonomously replicating sequence (ARS) elements that support the propagation of extrachromosomal plasmids. Budding yeast origins are ∼100 bp in size and share an A/T-rich ARS consensus sequence (ACS) that is essential for origin function. Six proteins that form the origin recognition complex (ORC) bind to the ACS in an ATP-dependent manner throughout the cell cycle (Bell and Stillman 1992). Beginning in late mitosis, Cdc6p interacts with origin-bound ORC to bring the six-protein MCM complex, a putative replicative helicase, onto the DNA (Aparicio et al. 1997; Perkins and Diffley 1997; Tanaka et al. 1997). In a late step requiring cyclin-dependent kinase (cdk) activity, Cdc45p is added to the prereplication complex, possibly as a scaffold component (Zou and Stillman 1999). Activation of another origin-associated kinase, Cdc7p, by its regulatory subunit Dbf4p, probably serves as the final signal activating replication fork movement (Dowell et al. 1994; Hardy 1998; Brown and Kelly 1999). By the time prereplication complexes fire, high cdk activity precludes the assembly of new complexes. Because a checkpoint ensures that the cell will not divide until replication is complete, and high levels of cdk activity are not reversed until mitosis is nearly finished, the genome will be replicated once and only once per cell cycle.

Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate.

To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural. While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E. coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction. The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model. We propose that the following sequential events may occur in both strains. A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally. this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication.

DnaA protein directs the binding of DnaB protein in initiation of DNA replication in Escherichia coli.

DnaA protein of Escherichia coli acts in the initiation of chromosomal replication to bind to sequences in the chromosomal origin. On binding, it promotes the assembly of other replication proteins that serve to prime DNA replication and assemble the replication apparatus for bidirectional replication fork movement. A collection of monoclonal antibodies to DnaA protein have been produced, one of which is described here, that interferes with the action of DnaA protein in promoting formation of a prepriming complex. On the analysis of this process, the antibody appears to interfere with the physical interaction between DnaA and DnaB protein in the DnaB.DnaC complex. Cross-linking studies confirm that DnaA and DnaB proteins interact directly. These results provide the first direct evidence that one of the roles of DnaA protein is to act as a site for binding of DnaB protein to the DNA and perhaps orients DnaB helicase to account for the directionality of replication fork movement.