Pre-replication Complex Formation Research Articles

DNA replication and progression through S phase

Initiation and completion of DNA replication defines the beginning and ending of S phase of the cell cycle. Successful progression through S phase requires that replication be properly regulated and monitored to ensure that the entire genome is duplicated exactly once, without errors, in a timely fashion. Given the immense size and complexity of eukaryotic genomes, this presents a significant challenge for the cell. As a result, DNA replication has evolved into a tightly regulated process involving the coordinated action of numerous factors that function in all phases of the cell cycle. We will review our current understanding of these processes from the formation of prereplicative complexes in preparation for S phase to the series of events that culminate in the loading of DNA polymerases during S phase. We will incorporate structural data from archaeal and bacterial replication proteins and discuss their implications for understanding the mechanism of action of their corresponding eukaryotic homologues. We will also describe the concept of replication licensing which protects against genomic instability by limiting initiation events to once per cell cycle. Lastly, we will review our knowledge of checkpoint pathways that maintain the integrity of stalled forks and relay defects in replication to the rest of the cell cycle.

Studies of the properties of human origin recognition complex and its Walker A motif mutants

The eukaryotic six-subunit origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. In this report we describe the isolation of the wild-type Homo sapiens (Hs)ORC and variants containing a Walker A motif mutation in the Orc1, Orc4, or Orc5 subunit using the baculovirus-expression system. Coexpression of all six HsORC subunits yielded a stable complex containing HsOrc subunits 1-5 (HsORC1-5) with virtually no Orc6 protein (Orc6p). We examined the ATPase, DNA-binding, and replication activities of these complexes. Similar to other eukaryotic ORCs, wild-type HsORC1-5 possesses ATPase activity that is stimulated only 2-fold by single-stranded DNA. HsORC1-5 with a mutated Walker A motif in Orc1p contains no ATPase activity, whereas a similar mutation of either the Orc4 or Orc5 subunit did not affect this activity. The DNA-binding activity of HsORC1-5, using lamin B2 DNA as substrate, is stimulated by ATP 3- to 5-fold. Mutations in the Walker A motif of Orc1p, Orc4p, or Orc5p reduced the binding efficiency of HsORC1-5 modestly (2- to 5-fold). Xenopus laevis ORC-depleted extracts supplemented with HsORC1-5 supported prereplication complex formation and X. laevis sperm DNA replication, whereas the complex with a mutation in the Walker A motif of the Orc1, Orc4, or Orc5 subunit did not. These studies indicate that the ATP-binding motifs of Orc1, Orc4, and Orc5 are all essential for the replication activity associated with HsORC.

Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis.

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. Here we describe a novel function of Geminin. We demonstrate that human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2.

Geminin Links Homeobox Domain Proteins to the Cell Cycle

Homeobox proteins play an essential role in pattern formation in development. Two groups report an antagonistic interaction between homeobox domain-containing proteins and the cell cycle regulator geminin. Geminin inhibits proliferation by binding to Cdt1, a protein required for the formation of prereplicative complexes needed for DNA replication. Del Bene et al. identified Xenopus geminin as an interacting partner of the homeobox domain-containing transcription factor Six3 in a yeast two-hybrid assay. Geminin binding to Six3 prevented Six3-dependent reporter gene activity but did not inhibit Six3 DNA binding. Six3 competed with Cdt1 for geminin binding, which may make Cdt1 available to participate in DNA replication and so allow cell proliferation. This activity appeared to be independent of the transcriptional regulatory activity of Six3. Various genetic assays confirmed the mutually antagonistic activities of Six3 for geminin's cell cycle suppression activity and geminin for Six3's proliferation activity. Luo et al. identified the Polycomb complex protein Scmh1 and various members of the Hox transcription factor family as partners of geminin in a yeast two-hybrid assay. Cultured cells exhibited colocalization of Polycomb complex proteins and geminin that was dependent on the phase of the cell cycle, with mitotic cells showing colocalization. Geminin also bound directly to DNA containing Hox regulatory elements and to the Hox proteins, which prevented their binding to DNA. Geminin inhibited Hox-stimulated reporter gene expression. Hox proteins also interact with Cdt1, and Hox and geminin competed for binding to Cdt1. These two papers link pathways controlling embryonic cell proliferation and pattern formation through geminin and suggest that not all transcription factor functions arise from regulation of gene expression (see Li and Rosenfeld for discussion). F. Del Bene, K. Tessmar-Raible, J. Wittbrodt, Direct interaction of geminin and Six3 in eye development. Nature 427 , 745-749 (2004). [Online Journal] L. Luo, X. Yang, Y. Takihara, H. Knoetgen, M. Kessel, The cell-cycle regulator geminin inhibits Hox function through direct and polycomb-mediated interactions. Nature 427 , 749-753 (2004). [Online Journal] X. Li, M. G. Rosenfeld, Origins of licensing control. Nature 427 , 687-688 (2004). [Online Journal]

Fission Yeast Cdc23/Mcm10 Functions after Pre-replicative Complex Formation To Promote Cdc45 Chromatin Binding

Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.

Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication.

Fission yeast Cut5/Rad4 and its budding yeast homolog Dpb11 are required for both DNA replication and the S-phase checkpoint. Here, we have investigated the role of the Xenopus homolog of Cut5 in the initiation of DNA replication using Xenopus egg extracts. Xenopus Cut5, which shows sequence similarity to DmMus101 and HsTopBP1, is essential for DNA replication in the egg extracts. It is required for the chromatin binding of Cdc45 and DNA polymerases, but not for the formation of pre-replicative complexes or the elongation stage of DNA replication. The chromatin binding of Cut5 consists of two distinct modes. S-phase cyclin-dependent kinase (S-CDK)-independent binding is sufficient for DNA replication while S-CDK-dependent binding is dispensable. Further, S-CDK acts after the chromatin binding of Cut5 and before the binding of Cdc45. These results demonstrate that the chromatin binding of Cut5 is required for the action of S-CDK, which in turn triggers the formation of pre-initiation complexes of DNA replication.

A Role for Ran-GTP and Crm1 in Blocking Re-Replication

All eukaryotic cells have regulatory mechanisms that limit genomic replication to a single round each cell cycle. These systems function by blocking formation of prereplication complexes. The regulatory mechanisms in the yeast S. cerevisiae have been identified, but these do not appear to be conserved in metazoans. Using Xenopus egg extracts, we have identified a metazoan-specific regulatory system that limits replication to a single round. We show that during S phase, soluble MCM helicase, an essential initiation factor, is inactivated when it associates with exportin-1/Crm1. Formation of this complex is dependent on both high Ran-GTP and cdk2 kinase activity. Lowering Ran-GTP within nuclei or nuclear extracts allows MCM to reassociate with chromatin during S phase and induces re-replication. Importantly, prevention of re-replication requires MCM-Crm1 complex formation, but it does not require export of MCM from the nucleus. Therefore, in metazoans, Crm1 functions in both nuclear export and blocking of re-replication.

Human cytomegalovirus infection leads to accumulation of geminin and inhibition of the licensing of cellular DNA replication.

Previous studies have shown that infection of G(0)-synchronized human fibroblasts by human cytomegalovirus (HCMV) results in a block to cellular DNA synthesis. In this study, we have examined the effect of viral infection on the formation of the host cell DNA prereplication complex (pre-RC). We found that the Cdc6 protein level was significantly upregulated in the virus-infected cells and that there was a delay in the expression of the Mcm family of proteins. The loading of the Mcm proteins onto the DNA pre-RC complex also appeared to be defective in the virus-infected cells. This inhibition of DNA replication licensing was associated with the accumulation of geminin, a replication inhibitor. Cdt1, which participates in the loading of the Mcm proteins, was also downregulated and modified differentially in the infected cells. Early viral gene expression was sufficient for the virus-induced alteration of the pre-RC, and the immediate-early protein IE1 was not required. These studies show that the inhibition of replication licensing in HCMV-infected cells is one of the multiple pathways by which the virus dysregulates the host cell cycle.

ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase.

The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks.

Essential role of human CDT1 in DNA replication and chromatin licensing.

Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication. A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA. In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein. We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells. We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin. Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle.

Identification of Tah11/Sid2 as the Ortholog of the Replication Licensing Factor Cdt1 in Saccharomyces cerevisiae

Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin-CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3). Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication. Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref. 10). Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.

The Human Origin Recognition Complex Protein 1 Dissociates from Chromatin during S Phase in HeLa Cells

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.

Multiple sites of replication initiation in the human beta-globin gene locus.

The cell cycle-dependent, ordered assembly of protein prereplicative complexes suggests that eukaryotic replication origins determine when genomic replication initiates. By comparison, the factors that determine where replication initiates relative to the sites of prereplicative complex formation are not known. In the human globin gene locus previous work showed that replication initiates at a single site 5' to the ss-globin gene when protein synthesis is inhibited by emetine. The present study has examined the pattern of initiation around the genetically defined ss-globin replicator in logarithmically growing HeLa cells, using two PCR-based nascent strand assays. In contrast to the pattern of initiation detected in emetine-treated cells, analysis of the short nascent strands at five positions spanning a 40 kb globin gene region shows that replication initiates at more than one site in non-drug-treated cells. Quantitation of nascent DNA chains confirmed that replication begins at several locations in this domain, including one near the initiation region (IR) identified in emetine-treated cells. However, the abundance of short nascent strands at another initiation site approximately 20 kb upstream is approximately 4-fold as great as that at the IR. The latter site abuts an early S phase replicating fragment previously defined at low resolution in logarithmically dividing cells.

Nucleosomes Positioned by ORC Facilitate the Initiation of DNA Replication

The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events. To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S. cerevisiae replication origins. Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function.

Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis.

Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation.

In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593-596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.

Cell cycle control as a basis for cancer drug development (Review).

Normal cell cycle progression relies on the cell's ability to translate extracellular signals such as mitogenic stimuli and intact extracellular matrices in order to efficiently replicate DNA and divide. Cyclin dependent kinases (cdks) respond to these signals and are largely responsible for positively pushing cells through the cell cycle. Due to their pivotal role in cell division, nature has evolved elaborate mechanisms to regulate the kinase activity of cdks. Cyclins are cdk binding partners which are required for kinase activity and their protein levels are intimately linked to the cell cycle stage. A variety of other cdk regulators such as phosphorylation events, natural inhibitors and complex stability are discussed. Phosphorylation of various cdk substrates results in diverse outcomes such as changes in gene expression, formation of prereplicative complexes and breakdown of the nuclear envelope. Cancer cells evolve in part by over-riding normal cell cycle regulation. Abnormal cdk activity is accomplished by cyclin amplification, cdk or substrate mutation as well as inactivation of inhibitors. The selective growth advantage of cancer cells also stems from amplification of positive growth signals, mutation of checkpoint and surveillance genes as well as deregulation of programmed cell death or apoptosis. The full potential of cancer therapies, such as small molecule inhibitors and gene therapy among others, focusing on our knowledge of cell cycle regulation has yet to be reached.

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis.

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.

Stepwise Regulated Chromatin Assembly of MCM2–7 Proteins

Acquisition of the competence to replicate requires the assembly of the MCM2-7 (minichromosome maintenance) protein complex onto pre-replicative chromatin, a step of the licensing reaction. This step is thought to occur through binding of a heterohexameric MCM complex containing the six related MCM subunits. Here we show that assembly of the MCM complex onto pre-replicative chromatin occurs through sequential stabilization of specific MCM subunits. Inhibition of licensing with 6-dimethylaminopurine results in chromatin containing specifically bound MCM4 and MCM6. A similar result was obtained by interference of the assembly reaction with an MCM3 antibody. The presence of chromatin-bound MCM intermediates was confirmed by reconstitution experiments in vitro with purified proteins and by the observation of an ordered association of MCM subunits with chromatin. These results indicate that the assembly of the MCM complex onto pre-replicative chromatin is regulated at the level of distinct subunits, suggesting an additional regulatory step in the formation of pre-replication complexes.