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.     

The MCM2-3-5 proteins: are they replication licensing factors?

DNA replication occurs only once in each normal mitotic cell cycle. To explain this strict control, a 'licensing factor' was proposed to enter the nucleus periodically as the nuclear envelope disintegrates and reassembles at the end of mitosis. Inactivation of licensing factor immediately following initiation of DNA synthesis would prevent reinitiation until after the next mitosis. The MCM2-3-5 proteins of Saccharomyces cerevisiae may be yeast's equivalent of licensing factor: they are present in the nucleus only between M and S phase, bind to chromatin and are important for the initiation of DNA replication.     

Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells

For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.     

MCM8 is an MCM2-7-related protein that functions as a DNA helicase during replication elongation and not initiation

MCM2-7 proteins are replication factors required to initiate DNA synthesis and are currently the best candidates for replicative helicases. We show that the MCM2-7-related protein MCM8 is required to efficiently replicate chromosomal DNA in Xenopus egg extracts. MCM8 does not associate with the soluble MCM2-7 complex and binds chromatin upon initiation of DNA synthesis. MCM8 depletion does not affect replication licensing or MCM3 loading but slows down DNA synthesis and reduces chromatin recruitment of RPA34 and DNA polymerase-alpha. Recombinant MCM8 displays both DNA helicase and ATPase activities in vitro. Reconstitution experiments show that ATP binding in MCM8 is required to rescue DNA synthesis in MCM8-depleted extracts. MCM8 colocalizes with replication foci and RPA34 on chromatin. We suggest that MCM8 functions in the elongation step of DNA replication as a helicase that facilitates the recruitment of RPA34 and stimulates the processivity of DNA polymerases at replication foci.     

Incremental Genetic Perturbations to MCM2-7 Expression and Subcellular Distribution Reveal Exquisite Sensitivity of Mice to DNA Replication Stress

Mutations causing replication stress can lead to genomic instability (GIN). In vitro studies have shown that drastic depletion of the MCM2-7 DNA replication licensing factors, which form the replicative helicase, can cause GIN and cell proliferation defects that are exacerbated under conditions of replication stress. To explore the effects of incrementally attenuated replication licensing in whole animals, we generated and analyzed the phenotypes of mice that were hemizygous for Mcm2, 3, 4, 6, and 7 null alleles, combinations thereof, and also in conjunction with the hypomorphic Mcm4^Chaos3 cancer susceptibility allele. Mcm4^{Chaos3/Chaos3} embryonic fibroblasts have ∼40% reduction in all MCM proteins, coincident with reduced Mcm2-7 mRNA. Further genetic reductions of Mcm2, 6, or 7 in this background caused various phenotypes including synthetic lethality, growth retardation, decreased cellular proliferation, GIN, and early onset cancer. Remarkably, heterozygosity for Mcm3 rescued many of these defects. Consistent with a role in MCM nuclear export possessed by the yeast Mcm3 ortholog, the phenotypic rescues correlated with increased chromatin-bound MCMs, and also higher levels of nuclear MCM2 during S phase. The genetic, molecular and phenotypic data demonstrate that relatively minor quantitative alterations of MCM expression, homeostasis or subcellular distribution can have diverse and serious consequences upon development and confer cancer susceptibility. The results support the notion that the normally high levels of MCMs in cells are needed not only for activating the basal set of replication origins, but also “backup” origins that are recruited in times of replication stress to ensure complete replication of the genome.     

Licensing of DNA replication by a multi-protein complex of MCM/P1 proteins in Xenopus eggs.

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re-association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.     

Schizosaccharomyces pombe minichromosome maintenance-binding protein (MCM-BP) antagonizes MCM helicase

The minichromosome maintenance (MCM) complex, a replicative helicase, is a heterohexamer essential for DNA duplication and genome stability. We identified Schizosaccharomyces pombe mcb1(+) (Mcm-binding protein 1), an apparent orthologue of the human MCM-binding protein that associates with a subset of MCM complex proteins. mcb1(+) is an essential gene. Deletion of mcb1(+) caused cell cycle arrest after several generations with a cdc phenotype and disrupted nuclear structure. Mcb1 is an abundant protein, constitutively present across the cell cycle. It is widely distributed in cytoplasm and nucleoplasm and bound to chromatin. Co-immunoprecipitation suggested that Mcb1 interacts robustly with Mcm3-7 but not Mcm2. Overproduction of Mcb1 disrupted the association of Mcm2 with other MCM proteins, resulting in inhibition of DNA replication, DNA damage, and activation of the checkpoint kinase Chk1. Thus, Mcb1 appears to antagonize the function of MCM helicase.     

Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation

Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae. We have cloned MCM10 from Drosophila melanogaster and show that it complements a ScMCM10 null mutant. Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2. Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1. RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content. Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation. The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants. Our results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked.     

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2～7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.     

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.     

Mcm3 is polyubiquitinated during mitosis before establishment of the pre-replication complex

To ensure fidelity in genome duplication, eukaryotes restrict DNA synthesis to once every cell division by a cascade of regulated steps. Central to this cascade is the periodic assembly of the hexameric MCM2-7 complex at replication origins. However, in Saccharomyces cerevisiae, only a fraction of each MCM protein is able to assemble into hexamers and associate with replication origins during M phase, suggesting that MCM complex assembly and recruitment may be regulated post-translationally. Here we show that a small fraction of Mcm3p is polyubiquitinated at the onset of MCM complex assembly. Reducing the rate of ubiquitination by uba1-165, a suppressor of mcm3-10, restored the interaction of Mcm3-10p with subunits of the MCM complex and its recruitment to the replication origin. Possible roles for ubiquitinated Mcm3p in the assembly of the MCM complex at replication origins are discussed.     

Bipolar spindle frequency and genome content are inversely regulated by the activity of two N-type kinesins in Entamoeba histolytica

Bipolar microtubular spindles are seen infrequently in Entamoeba histolytica trophozoites while monopolar or radial microtubular assemblies are common. Additionally, heterogeneity in nuclear DNA content and multi-nucleation is found in amoeba cells growing in axenic culture. Taken together these observations indicate that genome segregation is irregular in these cells. In order to identify proteins involved in regulating genome segregation, we have focused on studying E. histolytica homologues of kinesin motor proteins that are known to affect stability of bipolar mitotic spindles. We have demonstrated earlier that increased levels of the kinesin – Eh Klp5 – led to increased frequency of bipolar spindles accompanied with a reduction in the heterogeneity of genome content, showing that bipolar spindle frequency was inversely linked to genome content in E. histolytica . In this study, we have investigated the role of E. histolytica kinesins (Eh KlpA1, 2–4) in regulating bipolar spindle frequency and genome content. While downregulation of Eh Klp3, 4 and A1 showed no effect, downregulation of Eh Klp2 led to increased frequency of bipolar spindles and homogenization of genome content, similar to the effect of increased expression of Eh Klp5. In addition to microtubules, Eh Klp2–4 associated with F-actin in the cytoplasm, suggesting that these kinesins are multi-functional.     

A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.     

Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis.

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.     

MCM9 binds Cdt1 and is required for the assembly of prereplication complexes

Prereplication complexes (pre-RCs) define potential origins of DNA replication and allow the recruitment of the replicative DNA helicase MCM2-7. Here, we characterize MCM9, a member of the MCM2-8 family. We demonstrate that MCM9 binds to chromatin in an ORC-dependent manner and is required for the recruitment of the MCM2-7 helicase onto chromatin. Its depletion leads to a block in pre-RC assembly, as well as DNA replication inhibition. We show that MCM9 forms a stable complex with the licensing factor Cdt1, preventing an excess of geminin on chromatin during the licensing reaction. Our data suggest that MCM9 is an essential activating linker between Cdt1 and the MCM2-7 complex, required for loading the MCM2-7 helicase onto DNA replication origins. Thus, Cdt1, with its two opposing regulatory binding factors MCM9 and geminin, appears to be a major platform on the pre-RC to integrate cell-cycle signals.     

Characterization of Schizosaccharomyces pombe mcm7(+) and cdc23(+) (MCM10) and interactions with replication checkpoints

MCM proteins are required for the proper regulation of DNA replication. We cloned fission yeast mcm7(+) and showed it is essential for viability; spores lacking mcm7(+) begin S phase later than wild-type cells and arrest with an apparent 2C DNA content. We isolated a novel temperature-sensitive allele, mcm7-98, and also characterized two temperature-sensitive alleles of the fission yeast homolog of MCM10, cdc23(+). mcm7-98 and both cdc23ts alleles arrest with damaged chromosomes and an S phase delay. We find that mcm7-98 is synthetically lethal with the other mcmts mutants but does not interact genetically with either cdc23ts allele. However, cdc23-M36 interacts with mcm4ts. Unlike other mcm mutants or cdc23, mcm7-98 is synthetically lethal with checkpoint mutants Deltacds1, Deltachk1, or Deltarad3, suggesting chromosomal defects even at permissive temperature. Mcm7p is a nuclear protein throughout the cell cycle, and its localization is dependent on the other MCM proteins. Our data suggest that the Mcm3p-Mcm5p dimer interacts with the Mcm4p-Mcm6p-Mcm7p core complex through Mcm7p.     

Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.     

Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic. Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either replication licensing or S phase CDK activity. This revealed an unexpectedly broad system-wide effect on the chromatin proteome, indicating that the response to replication inhibition extends to many other functional modules in addition to the replication machinery. Several proteins that respond to replication inhibition (including nuclear pore proteins) coprecipitated with the Mcm2-7 licensing complex on chromatin, suggesting that Mcm2-7 play a central role in coordinating nuclear structure with DNA replication.     

Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae.

The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes. These patients are constitutively present in high abundance in actively proliferating cells. In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins. However, these proteins are limiting for the initiation of DNA synthesis at replication origins. Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase. The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle. We also show that S. cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle. These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.