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.     

Regulation of replication licensing by acetyltransferase Hbo1

The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.     

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.     

Activation of the replicative DNA helicase: breaking up is hard to do

The precise duplication of the eukaryotic genome is accomplished by carefully coordinating the loading and activation of the replicative DNA helicase so that each replication origin is unwound and assembles functional bi-directional replisomes just once in each cell cycle. The essential Minichromosome Maintenance 2-7 (Mcm2-7) proteins, comprising the core of the replicative DNA helicase, are first loaded at replication origins in an inactive form. The helicase is then activated by recruitment of the Cdc45 and GINS proteins into a holo-helicase known as CMG (Cdc45, Mcm2-7, GINS). These steps are regulated by multiple mechanisms to ensure that Mcm2-7 loading can only occur during G1 phase, whilst activation of Mcm2-7 cannot occur during G1 phase. Here we review recent progress in understanding these critical reactions focusing on the mechanism of helicase loading and activation.     

ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication

The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.     

MCM2–7 Proteins Are Essential Components of Prereplicative Complexes that Accumulate Cooperatively in the Nucleus during G1-phase and Are Required to Establish, But Not Maintain, the S-phase Checkpoint

A prereplicative complex (pre-RC) of proteins is assembled at budding yeast origins of DNA replication during the G1-phase of the cell cycle, as shown by genomic footprinting. The proteins responsible for this prereplicative footprint have yet to be identified but are likely to be involved in the earliest stages of the initiation step of chromosome replication. Here we show that MCM2-7 proteins are essential for both the formation and maintenance of the pre-RC footprint at the origin ARS305. It is likely that pre-RCs contain heteromeric complexes of MCM2-7 proteins, since degradation of Mcm2, 3, 6, or 7 during G1-phase, after pre-RC formation, causes loss of Mcm4 from the nucleus. It has been suggested that pre-RCs on unreplicated chromatin may generate a checkpoint signal that inhibits premature mitosis during S-phase. We show that, although mitosis does indeed occur in the absence of replication if MCM proteins are degraded during G1-phase, anaphase is prevented if MCMs are degraded during S-phase. Our data indicate that pre-RCs do not play a direct role in checkpoint control during chromosome replication.     

Dynamics of Pre-replicative Complex Assembly

The pre-replicative complex (pre-RC) is formed at all potential origins of replication through the action of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex. The end result of pre-RC formation is the loading of the Mcm2-7 replicative helicase onto origin DNA. We examined pre-RC formation in vitro and found that it proceeds through separable binding events. Origin-bound ORC recruits Cdc6, and this ternary complex then promotes helicase loading in the presence of a pre-formed Mcm2-7-Cdt1 complex. Using a stepwise pre-RC assembly assay, we investigated the fate of pre-RC components during later stages of the reaction. We determined that helicase loading is accompanied by dissociation of ORC, Cdc6, and Cdt1 from origin DNA. This dissociation requires ATP hydrolysis at a late stage of pre-RC assembly. Our results indicate that pre-RC formation is a dynamic process.     

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.     

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.     

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.     

The interaction networks of the budding yeast and human DNA replication-initiation proteins

DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G₁ transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.     

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.     

Analysis of Mcm2-7 chromatin binding during anaphase and in the transition to quiescence in fission yeast

Mcm2-7 proteins are generally considered to function as a heterohexameric complex, providing helicase activity for the elongation step of DNA replication. These proteins are loaded onto replication origins in M-G1 phase in a process termed licensing or pre-replicative complex formation. It is likely that Mcm2-7 proteins are loaded onto chromatin simultaneously as a pre-formed hexamer although some studies suggest that subcomplexes are recruited sequentially. To analyze this process in fission yeast, we have compared the levels and chromatin binding of Mcm2-7 proteins during the fission yeast cell cycle. Mcm subunits are present at approximately 1 x 10(4) molecules/cell and are bound with approximately equal stoichiometry on chromatin in G1/S phase cells. Using a single cell assay, we have correlated the timing of chromatin association of individual Mcm subunits with progression through mitosis. This showed that Mcm2, 4 and 7 associate with chromatin at about the same stage of anaphase, suggesting that licensing involves the simultaneous binding of these subunits. We also examined Mcm2-7 chromatin association when cells enter a G0-like quiescent state. Chromatin binding is lost in this transition in a process that does not require DNA replication or the selective degradation of specific subunits.     

1H, 15N and 13C chemical shift assignments of the Cdt1 binding domain of human Mcm6

The eukaryotic minichromsome maintenance (Mcm) proteins (Mcm2–7) are evolutionally conserved from yeast to human. These proteins are essential for DNA replication and Mcm6 is one subunit of Mcm2–7 complex that serves as the replicative helicase in DNA replication. Cdt1 is a critical member of pre-replicative complex (pre-RC), which directs the chromatin loading of Mcm2–7 complex. The Cdt1 binding domain (CBD) of human Mcm6 was found to directly interact with Cdt1 and this interaction may mediate the chromatin loading of Mcm2–7 complex. The structure of CBD exhibits a typical “winged-helix” fold which is generally involved in protein-nucleic acid interaction. Here we report the ¹H, ¹⁵N and ¹³C chemical shift assignments of human Mcm6 CBD determined by triple resonance experiments. The resonance assignments obtained in this work were required for the structure–function studies of CBD by NMR spectroscopy (BMRB deposits with accession number 16396).     

Cdt1 forms a complex with the minichromosome maintenance protein (MCM) and activates its helicase activity

Mcm4/6/7 forms a complex possessing DNA helicase activity, suggesting that Mcm may be a central component for the replicative helicase. Although Cdt1 is known to be essential for loading of Mcm onto the chromatin, its precise role in pre-RC formation and replication initiation is unknown. Using purified proteins, we show that Cdt1 forms a complex with Mcm4/6/7, Mcm2/3/4/5/6/7, and Mcm2/4/6/7 in glycerol gradient fractionation through interaction with Mcm2 and Mcm4/6. In the glycerol gradient fractionation, Mcm4/6/7-Cdt1 forms a complex (speculated to be a (Mcm4/6/7)2-Cdt13 assembly) in the presence of ATP, which is significantly larger than the Mcm4/6/7-Cdt1 complex generated in its absence. Furthermore, DNA binding and helicase activities of Mcm4/6/7 are significantly stimulated by Cdt1 protein in vitro. We generated a Cdt1 mutant, which fails to stimulate DNA binding and helicase activities of Mcm4/6/7. This mutant Cdt1 showed reduced interaction with Mcm and is deficient in the formation of a high molecular weight complex with Mcm. Thus, a productive interaction between Cdt1 and MCM appears to be essential for efficient loading of MCM onto template DNA, as well as for the efficient unwinding reaction.     

Multiple Cdt1 molecules act at each origin to load replication-competent Mcm2-7 helicases

Eukaryotic origins of replication are selected by loading a head-to-head double hexamer of the Mcm2-7 replicative helicase around origin DNA. Cdt1 plays an essential but transient role during this event; however, its mechanism of action is unknown. Through analysis of Cdt1 mutations, we demonstrate that Cdt1 performs multiple functions during helicase loading. The C-terminus of Cdt1 binds Mcm2-7, and this interaction is required for efficient origin recruitment of both proteins. We show that origin recognition complex (ORC) and Cdc6 recruit multiple Cdt1 molecules to the origin during helicase loading, and disruption of this multi-Cdt1 intermediate prevents helicase loading. Although dispensable for loading Mcm2-7 double hexamers that are topologically linked to DNA, the essential N-terminal domain of Cdt1 is required to load Mcm2-7 complexes that are competent for association with the Cdc45 and GINS helicase-activating proteins and replication initiation. Our data support a model in which origin-bound ORC and Cdc6 recruit two Cdt1 molecules to initiate double-hexamer formation prior to helicase loading and demonstrate that Cdt1 influences the replication competence of loaded Mcm2-7 helicases.     

Structural insights into the Cdt1-mediated MCM2-7 chromatin loading

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.     

Preventing re-replication of chromosomal DNA

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2-7 proteins to form a pre-replicative complex. Mcm2-7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.     

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.