Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase

Cdt1 is essential for loading Mcm2-7 proteins into prereplicative complexes (pre-RCs) during replication licensing and has been found in organisms as diverse as fission yeast and humans. We have identified a homologue of Cdt1 in Saccharomyces cerevisiae, which is required for pre-RC assembly. We show that, like Mcm2-7p, Cdt1p accumulates in the nucleus during G1 phase and is excluded from the nucleus later in the cell cycle by cyclin dependent kinases (cdks). Cdt1p interacts with the Mcm2--7p complex, and the nuclear accumulation of these proteins during G1 is interdependent. This coregulation of Cdt1p and Mcm2-7p represents a novel level of pre-RC control.     

Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.     

Essential role of MCM proteins in premeiotic DNA replication

A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2-7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation. The ability of mutants defective in MCM2-7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase. We show here that MCM2-7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle. This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication. Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication. Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles.     

Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases of the Clb/Cdc28 family restrict the initiation of DNA replication to once per cell cycle by preventing the re-assembly of pre-replicative complexes (pre-RCs) at replication origins that have already initiated replication. This assembly involves the Cdc6-dependent loading of six minichromosome maintenance (Mcm) proteins, Mcm2-7, onto origins. How Clb/Cdc28 kinases prevent pre-RC assembly is not understood. RESULTS: In living cells, the Mcm proteins were found to colocalize in a cell-cycle-regulated manner. Mcm2-4, 6 and 7 were concentrated in the nucleus in G1 phase, gradually exported to the cytoplasm during S phase, and excluded from the nucleus by G2 and M phase. Tagging any single Mcm protein with the SV40 nuclear localization signal made all Mcm proteins constitutively nuclear. In the absence of functional Cdc6, Clb/Cdc28 kinases were necessary and sufficient for efficient net nuclear export of a fusion protein between Mcm7 and the green fluorescent protein (Mcm7-GFP), whereas inactivation of these kinases at the end of mitosis coincided with the net nuclear import of Mcm7-GFP. In contrast, in the presence of functional Cdc6, which loads Mcm proteins onto chromatin, S-phase progression as well as Clb/Cdc28 kinases was required for Mcm-GFP export. CONCLUSIONS: We propose that Clb/Cdc28 kinases prevent pre-RC reassembly in part by promoting the net nuclear export of Mcm proteins. We further propose that Mcm proteins become refractory to this regulation when they load onto chromatin and must be dislodged by DNA replication before they can be exported. Such an arrangement could ensure that Mcm proteins complete their replication function before they are removed from the nucleus.     

Abundance of prereplicative complexes (Pre-RCs) facilitates recombinational repair under replication stress in fission yeast

Mcm2-7 complexes are loaded onto chromatin with the aid of Cdt1 and Cdc18/Cdc6 and form prereplicative complexes (pre-RCs) at multiple sites on each chromosome. Pre-RCs are essential for DNA replication and surviving replication stress. However, the mechanism by which pre-RCs contribute to surviving replication stress is largely unknown. Here, we isolated the fission yeast mcm6-S1 mutant that was hypersensitive to methyl methanesulfonate (MMS) and camptothecin (CPT), both of which cause forks to collapse. The mcm6-S1 mutation impaired the interaction with Cdt1 and decreased the binding of minichromosome maintenance (MCM) proteins to replication origins. Overexpression of Cdt1 restored MCM binding and suppressed the sensitivity to MMS and CPT, suggesting that the Cdt1-Mcm6 interaction is important for the assembly of pre-RCs and the repair of collapsed forks. MMS-induced Chk1 phosphorylation and Rad22/Rad52 focus formation occurred normally, whereas cells containing Rhp54/Rad54 foci, which are involved in DNA strand exchange and dissociation of the joint molecules, were increased. Remarkably, G(1) phase extension through deletion of an S phase cyclin, Cig2, as well as Cdt1 overexpression restored pre-RC assembly and suppressed Rhp54 accumulation. A cdc18 mutation also caused hypersensitivity to MMS and CPT and accumulation of Rhp54 foci. These data suggest that an abundance of pre-RCs facilitates a late step in the recombinational repair of collapsed forks in the following S phase.     

Pre-replicative complex assembly with purified proteins

Licensing of origins of eukaryotic DNA replication involves the loading of six minichromosome maintenance proteins (Mcm2-7) into pre-replicative complexes (pre-RCs). The assembly of the pre-RC is restricted to G1 phase of the cell cycle, which is crucial to ensure once per cell cycle DNA replication. Mcm2-7 is loaded by the action of the origin recognition complex (ORC), Cdc6 and Cdt1 and requires ATP. In vitro reconstitution of this reaction has shown that Mcm2-7 is loaded onto DNA as a symmetrical head-to-head double hexamer. We describe in detail how pre-RC proteins are purified and used to reconstitute pre-RC formation in vitro. This method is useful for studying the biochemical mechanisms of Mcm2-7 loading as well as subsequent steps in DNA replication.     

Activation of a human chromosomal replication origin by protein tethering

The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4^DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences.     

Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression

Origins of DNA replication are licensed in G1 by recruiting the minichromosome maintenance (MCM) proteins to form a prereplicative complex (pre-RC). Prior to initiation of DNA synthesis from each origin, a preinitiation complex (pre-IC) containing Cdc45 and other proteins is formed. We report that Cdc7-Dbf4 protein kinase (DDK) promotes assembly of a stable Cdc45-MCM complex exclusively on chromatin in S phase. In this complex, Mcm4 is hyperphosphorylated. Studies in vitro using purified DDK and Mcm4 demonstrate that hyperphosphorylation occurs at the Mcm4 N terminus. However, the DDK substrate specificity is conferred by an adjacent DDK-docking domain (DDD), sufficient for facilitating efficient phosphorylation of artificial phosphoacceptors in cis. Genetic evidence suggests that phosphorylation of Mcm4 by DDK is important for timely S phase progression and for cell viability upon overproduction of Cdc45. We suggest that DDK docks on and phosphorylates MCM proteins at licensed origins to promote proper assembly of pre-IC.     

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G₁ phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G₁ phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G₁ phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC.     

Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability

The minichromosome maintenance (MCM) complex plays essential, conserved roles throughout DNA synthesis: first, as a component of the prereplication complex at origins and, then, as a helicase associated with replication forks. Here we use fission yeast (Schizosaccharomyces pombe) as a model to demonstrate a role for the MCM complex in protecting replication fork structure and promoting recovery from replication arrest. Loss of MCM function generates lethal double-strand breaks at sites of DNA synthesis during replication elongation, suggesting replication fork collapse. MCM function also maintains the stability of forks stalled by hydroxyurea that activate the replication checkpoint. In cells where the checkpoint is activated, Mcm4 binds the Cds1 kinase and undergoes Cds1-dependent phosphorylation. MCM proteins also interact with proteins involved in homologous recombination, which promotes recovery from arrest by ensuring normal mitosis. We suggest that the MCM complex links replication fork stabilization with checkpoint arrest and recovery through direct interactions with checkpoint and recombination proteins and that this role in S-phase genome stability is conserved from yeast to human cells.     

Chromosomal Mcm2-7 distribution and the genome replication program in species from yeast to humans

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.     

CDK phosphorylation of a novel NLS-NES module distributed between two subunits of the Mcm2-7 complex prevents chromosomal rereplication

Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.     

Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, approximately 30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins.     

A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase

During G1-phase of the cell-cycle the replicative MCM2–7 helicase becomes loaded onto DNA into pre-replicative complexes (pre-RCs), resulting in MCM2–7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number of helicase-activation factors the assembly of a Cdc45–MCM2–7–GINS (CMG) complex. However, in the absence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2–7 double-hexamer represents a very large interaction surface. Currently it is unclear what mechanisms restricts complex assembly and how DDK can overcome this inhibition to promote CMG-assembly. We developed an advanced reconstituted-system to study helicase activation in-solution and discovered that individual factors like Sld3 and Sld2 can bind directly to the pre-RC, while Cdc45 cannot. When Sld3 and Sld2 were incubated together with the pre-RC, we observed that competitive interactions restrict complex assembly. DDK stabilizes the Sld3/Sld2–pre-RC complex, but the complex is only short-lived, indicating an anti-cooperative mechanism. Yet, a Sld3/Cdc45–pre-RC can form in the presence of DDK and the addition of Sld2 enhances complex stability. Our results indicate that helicase activation is regulated by competitive and cooperative interactions, which restrict illegitimate complex formation and direct limiting helicase-activation factors into pre-initiation complexes.     

Is There A Pre-RC Checkpoint That Cancer Cells Lack?

Critical for genomic integrity, accurate DNA replication is tightly regulated by the convergence of prereplication protein complexes (pre-RCs) to “license” replicating origins on DNA in G1 and is activated by S-phase promoting kinases that selectively target and trigger origin firing in S-phase. To present, a checkpoint mechanism monitoring pre-RC complex formation and activation has yet to be elucidated. However, perturbation of these protein complexes has yielded divergent phenotypes in recent reports: normal cells arrest in the cell cycle, whereas cancerous cells arrest and die. These data implicate a mechanism by which normal cells sense pre-RC deficiency and then signal for cell cycle arrest. The potential for therapeutic exploits of this disparity between normal and cancer cells is apparent. Here, we explore recent data supporting the existence of a pre-RC checkpoint that ensures faithful pre-RC formation, a cell cycle mechanism that is intriguingly compromised in cancer cells.     

Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase

Loading of the Mcm2-7 DNA replicative helicase onto origin-proximal DNA is a critical and tightly regulated event during the initiation of eukaryotic DNA replication. The resulting protein-DNA assembly is called the prereplicative complex (pre-RC), and its formation requires the origin recognition complex (ORC), Cdc6, Cdt1, and ATP. ATP hydrolysis by ORC is required for multiple rounds of Mcm2-7 loading. Here, we investigate the role of ATP hydrolysis by Cdc6 during pre-RC assembly. We find that Cdc6 is an ORC- and origin DNA-dependent ATPase that functions at a step preceding ATP hydrolysis by ORC. Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 on origin DNA and prevents Mcm2-7 loading. In contrast, the initial association of Mcm2-7 with the other pre-RC components does not require ATP hydrolysis by Cdc6. Importantly, these coordinated yet distinct functions of ORC and Cdc6 ensure the correct temporal and spatial regulation of pre-RC formation.     

Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway.

Eukaryotic cells coordinate chromosome duplication by assembly of protein complexes at origins of DNA replication and by activation of cyclin-dependent kinase and Cdc7p-Dbf4p kinase. We show in Saccharomyces cerevisiae that although Cdc7p levels are constant during the cell division cycle, Dbf4p and Cdc7p-Dbf4p kinase activity fluctuate. Dbf4p binds to chromatin near the G(1)/S-phase boundary well after binding of the minichromosome maintenance (Mcm) proteins, and it is stabilized at the non-permissive temperature in mutants of the anaphase-promoting complex, suggesting that Dbf4p is targeted for destruction by ubiquitin-mediated proteolysis. Arresting cells with hydroxyurea (HU) or with mutations in genes encoding DNA replication proteins results in a more stable, hyper-phosphorylated form of Dbf4p and an attenuated kinase activity. The Dbf4p phosphorylation in response to HU is RAD53 dependent. This suggests that an S-phase checkpoint function regulates Cdc7p-Dbf4p kinase activity. Cdc7p may also play a role in adapting from the checkpoint response since deletion of CDC7 results in HU hypersensitivity. Recombinant Cdc7p-Dbf4p kinase was purified and both subunits were autophosphorylated. Cdc7p-Dbf4p efficiently phosphorylates several proteins that are required for the initiation of DNA replication, including five of the six Mcm proteins and the p180 subunit of DNA polymerase alpha-primase.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45

Current models suggest that the replication initiation factor Mcm10 is required for association of Mcm2-7 with origins of replication to generate the prereplicative complex (pre-RC). Here we report that Xenopus Mcm10 (XMcm10) is not required for origin binding of XMcm2-7. Instead, the chromatin binding of XMcm10 at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7. In the absence of XMcm10, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked. Therefore, XMcm10 performs its function after pre-RC assembly and before origin unwinding. As one of the earliest known pre-RC activation steps, chromatin binding of XMcm10 is an attractive target for regulation by cell cycle checkpoints.