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Results for “Minichromosome Maintenance Complex Component 7”

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PubMed2002

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.

Amino Acid Sequence↗

PubMed2004

A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication.

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.

Animals↗

PubMed2004

Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling.

Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.

Aphidicolin↗

PubMed2005

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.

Adenosine Triphosphatases↗

PubMed2003

Human cytomegalovirus prevents replication licensing by inhibiting MCM loading onto chromatin.

To allow DNA replication only once per cell cycle, origins of replication are reactivated ('licensed') during each G1 phase. Licensing is facilitated by assembly of the pre-replicative complex (pre-RC) at origins that concludes with loading the mini-chromosome maintenance (MCM) complex onto chromatin. Here we show that a virus exploits pre-RC assembly to selectively inhibit cellular DNA replication. Infection of quiescent primary fibroblasts with human cytomegalovirus (HCMV) induces all pre-RC factors. Although this is sufficient to assemble the MCM-loading factors onto chromatin, as it is in serum-stimulated cells, the virus inhibits loading of the MCM complex itself, thereby prematurely abrogating replication licensing. This provides a new level of control in pre-RC assembly and a mechanistic rationale for the unusual HCMV-induced G1 arrest that occurs despite the activation of the cyclin E-dependent transcription programme. Thus, this particularly large virus might thereby secure the supply with essential replication factors but omit competitive cellular DNA replication.

Animals↗

PubMed2002

Cell type-specific responses of human cells to inhibition of replication licensing.

Replication origins are 'licensed' for a single initiation event by loading Mcm2-7 complexes during late mitosis and G1. Licensing is blocked at other cell cycle stages by the activity of cyclin-dependent kinases and a small protein called geminin. Here, we describe the effects of over-expressing a non-degradable form of geminin in various cell lines. Geminin expression reduced the quantity of Mcm2 bound to chromatin and blocked cell proliferation. U2OS (p53+/Rb+) cells showed an early S phase arrest with high cyclin E and undetectable cyclin A levels, consistent with the activation of an intra-S checkpoint. Saos2 (p53-/Rb-) cells showed an accumulation of cells in late S and G2/M with approximately normal levels of cyclin A, consistent with loss of this intra-S phase checkpoint. Geminin also induced apoptosis in both these cell lines. In contrast, IMR90 primary fibroblasts over-expressing geminin arrested in G1 with reduced cyclin E levels and no detectable apoptosis. A 'licensing checkpoint' may therefore act in primary cells to prevent passage into S phase in the absence of sufficient origin licensing. These results suggest that inhibition of the licensing system may cause cancer-specific cell killing and therefore represent a novel anti-cancer target.

Adenoviridae↗

PubMed2004

Regulation of Geminin and Cdt1 expression by E2F transcription factors.

Geminin and Cdt1 play an essential role in the initiation of DNA replication, by regulating the chromatin loading of the MCM complex. In this study, we showed that the transcription of human Geminin and Cdt1, as well as that of MCM7, is activated by transcription factors E2F1-4, but not by factors E2F5-7. Analysis of various Geminin and Cdt1 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the transcriptional activation. The promoter activity for human Geminin was activated by the E7, but not E6, oncogene of human papillomavirus type 16. While E2F1-induced activation of human Cdt1 gene transcription was suppressed by pRb, but not by p107 or p130, its E2F4-induced activation was suppressed by pRb, p107, and p130. Furthermore, the promoter activities of human Geminin and Cdt1 were demonstrated to be growth-dependent. Taken together, the results demonstrate that Geminin and Cdt1 constitute targets for various members of the E2F family of transcription factors, and that expression of Geminin and Cdt1 is perhaps mediated by the activation of a pRb/E2F pathway.

Base Sequence↗

PubMed2006

MCM7 amplification and overexpression are associated with prostate cancer progression.

The genomic DNA profiles of prostate cancers with aggressive features were compared to the profiles of matched normal DNA to identify genes that are selectively amplified in the cancer cells. One of the identified genes, MCM7, which is a component of the DNA replication licensing complex, has been studied extensively both at the DNA and protein levels in human prostate tissues. Approximately half of the prostate cancer specimens studied showed MCM7 gene amplification, and 60% of the aggressive prostate cancer specimens had increased MCM7 protein expression. Amplification or overexpression of MCM7 was significantly associated with relapse, local invasion and a worse tumor grade. Constitutive expression of MCM7 in a human prostate cancer cell line, DU145, resulted in markedly increased DNA synthesis and cell proliferation compared to vector-only controls, and an increased cell invasion in vitro. Indeed, MCM7 overexpression produced primary tumors 12 times larger than vector-only controls and resulted in a rapid demise of mice bearing those tumors. These studies implicate MCM7, and the DNA replication licensing gene family, in prostate cancer progression, growth and invasion.

Animals↗

PubMed2003

Mammalian Mcm2/4/6/7 complex forms a toroidal structure.

BACKGROUND: The Mcm proteins are a family of six homologous proteins (Mcm2-7) that play an important role in DNA replication. They form Mcm4/6/7 and Mcm2/4/6/7 complexes, but their structures are not known. RESULTS: We found that the human Mcm2/4/6/7 tetramer forms a toroidal structure, with a central cavity about 3-4 nm in diameter. Observations were made using electron microscopy, employing the image analysis of single particles. The most predominant averaged image displayed a toroid harbouring four bulges forming corners, one of which was larger than the others. This structure was very similar to the mouse Mcm2/4/6/7 tetramer that was independently prepared and analysed by electron microscopy. These toroidal structures are distinct from that of the Mcm4/6/7 hexamer, which was also examined by electron microscopy. GST(glutathione S-transferase)-pull down and two hybrid experiments suggest that a putative Mcm6-Mcm6 hinge contributes to the formation of the Mcm7/4/6/6/4/7 heterohexamer. CONCLUSIONS: The Mcm2/4/6/7 tetramer forms a toroidal structure that is distinct from that of the Mcm4/6/7 hexamer in size and shape.

Animals↗

PubMed2004

Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases.

The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. We describe four lines of evidence that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex. First, ATM phosphorylates MCM3 on S535 in response to ionizing radiation. Second, ATR phosphorylates MCM2 on S108 in response to multiple forms of DNA damage and stalling of replication forks. Third, ATR-interacting protein (ATRIP)-ATR interacts with MCM7. Fourth, reducing the amount of MCM7 in cells disrupts checkpoint signaling and causes an intra-S-phase checkpoint defect. Thus, the MCM complex is a platform for multiple DNA damage-dependent regulatory signals that control DNA replication.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1995

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

Amino Acid Sequence↗

PubMed1996

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

Animals↗

PubMed1996

Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.

We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect. Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins. We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p. These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes. Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1.

Amino Acid Sequence↗

PubMed1996

Binding of human minichromosome maintenance proteins with histone H3.

Minichromosome maintenance (MCM) proteins play essential roles in eukaryotic DNA replication, but their biochemical properties remain to be determined. We detected in HeLa cell extracts six proteins, CDC47, CDC46/MCM5, Cdc21, P1/MCM3, Mis5, and BM28/MCM2, by their binding to a specific antibody and by partial sequencing. The human homologs of the MCM2 (BM28), Mis5, Cdc21, and CDC47 proteins were tightly bound to a histone-Sepharose column and purified to near homogeneity, whereas the P1/MCM3 and CDC46/MCM5 proteins passed through. Among the four core histones, the human BM28/MCM2, Mis5, Cdc21, and CDC47 proteins had high affinity for histone H3. Immunoprecipitation with anti-Cdc21 antibody revealed that these four MCM proteins form complexes. These results are consistent with the findings that MCM proteins bind with chromatin in vivo.

Amino Acid Sequence↗

PubMed1996

hCDC47, a human member of the MCM family. Dissociation of the nucleus-bound form during S phase.

hCDC47 is a putative human homologue of yeast CDC47 and a member of the MCM protein family, which has been implicated in the regulatory machinery causing DNA to replicate only once in the S phase. In the present study, we performed an initial characterization of hCDC47. We found that hCDC47 protein was present in the nucleus of cultured human cells in two different forms; one extractable by a non-ionic detergent and the other resistant to such extraction and tightly associated with the nucleus. The levels of the nucleus-bound form gradually diminished during S phase progression, although the total amount of nuclear hCDC47 protein remained relatively constant, suggesting that the nucleus-bound form becomes dissociated from the nuclear structure during DNA replication. This behavior of hCDC47 protein is very similar to that of other mammalian MCM proteins reported recently. We also found that expression of hCDC47 mRNA was repressed in quiescent cells but was induced at the late G1 to S phase by growth factor stimulation. Together, these findings indicate that hCDC47 protein together with other MCM proteins participates in the regulation of mammalian DNA replication.

Cell Cycle↗

PubMed1997

In vivo interaction of human MCM heterohexameric complexes with chromatin. Possible involvement of ATP.

The MCM protein family, which consists of at least six members, has been implicated in the regulatory machinery causing DNA to replicate once in the S phase. Mammalian MCM proteins are present in the nucleus in two different forms, one extractable by nonionic detergents and the other resistant to such extraction. The latter is assumed to be tightly associated with nuclear structures and released at the time of initiation of replication. However, details of the mode of binding remain unclear. In the present study, we found that, in nonionic detergent-permeabilized nuclei, the association of human MCM (hMCM) proteins with them could be stabilized by the addition of ATP. The hMCMs bound to the nuclei in the presence of ATP were released by digestion with nucleases, suggesting that they are chromatin-associated. The nuclease-directed solubilization of the chromatin-bound hMCMs thus provided a means to analyze them as well as soluble hMCMs by co-immunoprecipitation. The results indicate that the six hMCM members exist as heterocomplexes, whether bound or unbound. We therefore propose that hMCM proteins may function in DNA replication as heterohexamers associated with chromatin and that ATP is possibly involved in the association. Nuclease digestion-immunoprecipitation techniques of the type described here should facilitate further elucidation of the mode of interaction between hMCMs and chromatin.

Adenosine Triphosphate↗

PubMed1997

A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex.

All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3' to 5' direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.

Adenosine Triphosphatases↗

PubMed1998

MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells.

In a search for new partners of the activated form of Rac GTPase, we have isolated through a two-hybrid cloning procedure a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family GTPases. A specific mRNA of 3.2 kilobases was detected in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which we call MgcRacGAP, was detected in human testis as well as in germ cell tumor extracts by immunoblotting with antibodies specific to recombinant protein. In vitro, the GAP domain of MgcRacGAP strongly stimulates Rac1 and Cdc42 GTPase activity but is almost inactive on RhoA. N-terminal to its GAP domain, MgcRacGAP contains a cysteine-rich zinc finger-like motif characteristic of the Chimaerin family of RhoGAPs. The closest homolog of MgcRacGAP is RotundRacGAP, a product of the Drosophila rotund locus. In situ hybridization experiments in human testis demonstrate a specific expression of mgcRacGAP mRNA in spermatocytes similar to that of rotundRacGAP in Drosophila testis. Therefore, protein sequence similarity and analogous developmental and tissue specificities of gene expression support the hypothesis that RotundRacGAP and MgcRacGAP have equivalent functions in insect and mammalian germ cells. Since rotundRacGAP deletion leads to male sterility in the fruit fly, the mgcRacGAP gene may prove likewise to play a key role in mammalian male fertility.

Amino Acid Sequence↗