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.     

REC, Drosophila MCM8, drives formation of meiotic crossovers

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2–7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products.     

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.     

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica.     

Ancient diversification of eukaryotic MCM DNA replication proteins

Background  

Yeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7) for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear.     

Minichromosome maintenance protein 5 homologue in Caenorhabditis elegans plays essential role for postembryonic development

Genome duplication is tightly controlled in multicellular organisms to ensure the genome stability. Studies in Saccharomyces cerevisiae and Xenopus show that minichromosome maintenance (MCM) proteins are essential for genome duplication. However, the development role of MCM proteins in multicellular organisms is not well known. MCM5 encodes a member of the MCM2-7 protein family involved in the initiation of DNA replication. The sequences of all Mcm5 homologues from yeast to human are highly conserved and suggest that their functions are also conserved. Here, we isolated the first mutant allele of mcm-5 (fw7) in Caenorhabditis elegans. Homozygous mcm-5 (fw7) mutants from heterozygous parents exhibited variable larval lethality and adult sterility. The postembryonically born neuron number was decreased and also showed aberrant axon morphology. Our study revealed that the losses of neurons in mcm-5 (fw7) mutants were caused by cell cycle defects not by programmed cell death. The examination showed that mcm-5 was widely used for postembryonic development in multiple cells such as seam cells, gonad and intestinal cells. Knockdown of mcm-5 by RNAi caused 98.1% embryonic arrest, suggesting that mcm-5 was also required for embryonic development. After RNAi treatment of the other MCM2-7 family members, we found that they all exhibited similar phenotypes as mcm-5, suggesting that the MCM2-7 family in C. elegans might function associated with cell division as its homologues in S. cerevisiae.     

The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding

Eukaryotes have six minichromosome maintenance (MCM) proteins that are essential for DNA replication. The contribution of ATPase activity of MCM complexes to their function in replication is poorly understood. We have established a cell-free system competent for replication in which all MCM proteins are supplied by purified recombinant Xenopus MCM complexes. Recombinant MCM2-7 complex was able to assemble onto chromatin, load Cdc45 onto chromatin, and restore DNA replication in MCM-depleted extracts. Using mutational analysis in the Walker A motif of MCM6 and MCM7 of MCM2-7, we show that ATP binding and/or hydrolysis by MCM proteins is dispensable for chromatin loading and pre-replicative complex (pre-RC) assembly, but is required for origin unwinding during DNA replication. Moreover, this ATPase-deficient mutant complex did not support DNA replication in MCM-depleted extracts. Altogether, these results both demonstrate the ability of recombinant MCM proteins to perform all replication roles of MCM complexes, and further support the model that MCM2-7 is the replicative helicase. These data establish that mutations affecting the ATPase activity of the MCM complex uncouple its role in pre-RC assembly from DNA replication.     

REC, a new member of the MCM-related protein family, is required for meiotic recombination in Drosophila

rec mutations result in an extremely low level of recombination and a high frequency of primary non-disjunction in the female meiosis of Drosophila melanogaster. Here we demonstrate that the rec gene encodes a novel protein related to the mini-chromosome maintenance (MCM) proteins. Six MCM proteins (MCM2-7) are conserved in eukaryotic genomes, and they function as heterohexamers in the initiation and progression of mitotic DNA replication. Three rec alleles, rec(1), rec(2) and rec (3), were found to possess mutations within this gene, and P element-mediated germline transformation with a wild-type rec cDNA fully rescued the rec mutant phenotypes. The 885 amino acid REC protein has an MCM domain in the middle of its sequence and, like MCM2, 4, 6 and 7, REC contains a putative Zn-finger motif. Phylogenetic analyses revealed that REC is distantly related to the six conserved MCM proteins. Database searches reveal that there are candidates for orthologs of REC in other higher eukaryotes, including human. We addressed whether rec is involved in DNA repair in the mitotic division after the DNA damage caused by methylmethane sulfonate (MMS) or by X-rays. These analyses suggest that the rec gene has no, or only a minor, role in DNA repair and recombination in somatic cells.     

Unwinding to recombine

The MCM proteins are best known for their role in DNA replication, MCM2-7 forming the replicative helicase. Now, two reports in this issue of Molecular Cell, Nishimura et?al. (2012) and Lutzmann et?al. (2012) show the less well understood MCM8 and MCM9 to be crucial for effective homologous recombination.     

MCM proteins: DNA damage, mutagenesis and repair

The MCM2-7 complex, which may act as a replicative helicase during DNA synthesis, plays a central role in S-phase genome stability. MCM proteins are required for processive DNA replication and are a target of S-phase checkpoints. Loss of MCM function causes DNA damage and genome instability. MCM expression is upregulated in proliferating cells, providing a diagnostic marker for both cancerous cells and cells with the potential to become malignant. The role of the MCM complex in genome integrity reflects its activity both at active replication forks and away from forks.     

Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins

The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.     

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2–7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.DNA replication during the S phase necessitates that the entire genome be duplicated with the minimum of errors. Thousands of replication forks are involved in this process and they must be coordinated to ensure that every section of DNA is only replicated once. Errors in DNA replication are likely to be a major cause of the genetic instability that can lead to cancer (1). Cells are able to prevent duplicate replication of DNA by having a distinct stage that occurs during the G1 phase when replication origins are “licensed” for replication, a process that involves the preloading of several proteins involved in DNA replication (2). As DNA is replicated at each origin, these proteins are removed, thereby ensuring that each origin fires only once during each S phase. DNA damage response kinases activated by the stalled forks prevent the replication machinery from being activated in new chromosome domains, indicating a tight relationship between the DNA damage response and the DNA replication pathways (3, 4).The first step of the replication licensing mechanism is the loading of the minichromosome maintenance (MCM)¹ proteins on to replication origins along with origin recognition complex proteins, Cdt6 and Cdt1 (5). The eukaryotic MCM complex consists of six paralogs that form a heterohexameric ring. All eukaryotic organisms possess six homologous proteins (MCM2-MCM7) that form a heterohexameric ring that belong to the family of AAA+ (ATPase associated with various cellular activities) proteins and share similarities to other hexameric helicases (6). Even though additional MCM proteins have been identified in higher eukaryotes, the MCM2-MCM7 complex remains the prime candidate for the role of replicative helicase (7). MCM2–7 is required for both initiation and elongation of DNA replication, with its regulation at each stage being an essential player of eukaryotic DNA replication (8). As a critical mechanism to ensure only a single round of DNA replication, the loading of additional MCM2–7 complexes onto origins of replication is inactivated by redundant mechanisms after passage into S phase (9).The MCM complex plays a crucial role in determining the replication potential of cells, but recent work suggests that MCM proteins are not only targets of the S-phase checkpoints, but they also interact directly with components of the checkpoint and repair pathways (10, 11). In yeast, temperature sensitive MCM cells at restrictive temperature contain numerous foci recognized by the phosphorylated histone H2AX antibody (12), suggesting a role in the repair of DNA double-strand breaks. Although, in principle, only two DNA helicase activities are required to establish a bidirectional replication fork from each origin, a relatively large excess of MCM complexes are loaded at origins of replication and distributed along the chromatin (13). Their function is not well understood, and most of them are displaced from the DNA during S-phase, apparently without having played an active role in DNA replication. The “MCM paradox” refers to the fact that, at least in yeast, Xenopus, Drosophila, and mammalian cells, it is possible to reduce the concentration of MCM proteins by more than 90% without impairing DNA replication (14–18) and also refers to the observation that the majority of MCM complexes do not localize to the sites of DNA synthesis in mammalian cells, further suggesting a potential role for the MCM proteins beyond DNA replication.Using a combination of stable isotope labeling with amino acids in cell culture (SILAC)–based quantitative proteomics (19) with immunoprecipitation of green fluorescent protein (GFP)-tagged fusion proteins (20), we identified differences in protein binding partners with the MCM complex following DNA damage. Stable cell lines expressing GFP-tagged MCM2 and MCM5 were used in immunoprecipitation experiments from cells that were either mock treated, or treated with Etoposide for 15, 60, and 240 min. Etoposide is an antitumor drug that stabilizes a covalent complex between the DNA topoisomerase II and DNA by interfering with the cleavage-ligation reaction of the topoisomerase (21). This revealed specific interaction between the MCM complex and several proteins such as Nucleophosmin, BAG2, UPP1, and HDAC10. Interestingly, the MCM complex showed dynamic changes in interaction with Importin7 and the histone chaperone ASF1, and a decrease in interaction with the Chromodomain helicase DNA binding protein 3 (CHD3) resulting from the treatment with etoposide. This increase in interaction with ASF1 was followed by an enrichment of histone proteins, suggesting a novel role for the MCM proteins in histone deposition on chromatin following DNA damage.     

Interactions of the human MCM-BP protein with MCM complex components and Dbf4

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.     

Minichromosome maintenance as a genetic assay for defects in DNA replication.

Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication. Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs. The ARS-specific MCM genes are essential for the initiation of DNA replication. Among these are members of the MCM2-7 family that encode subunits of the preinitiation complex and MCM10, whose gene product interacts with members of the Mcm2-7 proteins. Among the ARS-nonspecific MCM gene products are chromosome transmission factors. Refinement of this genetic assay as a screening tool and further analysis of existing mcm mutants may reveal new replication initiation proteins.     

Drosophila MCM protein complexes.

MCM genes encode a family of evolutionarily conserved proteins required for DNA replication. In Saccharomyces cerevisiae, where they were first identified, MCM genes interact genetically with each other. Allele specificity in these interactions suggests that MCM proteins physically associate with one another and that this association is essential for function. We describe here an analysis of physical interactions among three Drosophila MCM proteins. Using specific antibodies we detect Drosophila MCMs almost exclusively in 600-kDa protein complexes. Co-immunoprecipitation data demonstrate the existence of at least two distinct types of 600-kDa complexes, one that contains DmCDC46 and one that appears to contain both DmMCM2 and Dpa (a CDC54 homologue). These complexes are stable throughout embryonic division cycles, are resistant to treatments with salt and detergent, and are present during development in tissues undergoing mitotic DNA replication as well as endoreplication. When extracts are prepared under low salt conditions all three MCM proteins co-immunoprecipitate. Consequently, we suggest that the 600-kDa complexes interact in a higher order complex.