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Results for “Minichromosome Maintenance Complex Component 7”

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At least 19 recordsLinked to original sources

PubMed2005

Involvement of human MCM8 in prereplication complex assembly by recruiting hcdc6 to chromatin.

The MCM2-MCM7 complex is an essential component of the prereplication complex (pre-RC), which is recruited by the cdc6 and cdt1 proteins to origins of DNA replication during G(1) phase. Here, we report that the accumulation on chromatin of another member of the MCM protein family, human MCM8 (hMCM8), occurs during early G(1) phase, before the hMCM2-hMCM7 complex binds. hMCM8 interacts in vivo with two components of the pre-RC, namely, hcdc6 and hORC2. Depletion of endogenous hMCM8 protein by RNA interference leads to a delay of entry into S phase, suggesting a role for hMCM8 in G(1) progression. Furthermore, down-regulation of hMCM8 also leads to a reduced loading of hcdc6 and the hMCM2-hMCM7 complex on chromatin. These results suggest that hMCM8 is a crucial component of the pre-RC and that the interaction between hMCM8 and hcdc6 is required for pre-RC assembly.

Cell Cycle Proteins↗

PubMed2001

Initiating DNA synthesis: from recruiting to activating the MCM complex.

The exact duplication of a genome once per cell division is required of every proliferating cell. To achieve this goal, eukaryotes adopt a strategy that limits every replication origin to a single initiation event within a narrow window of the cell cycle by temporally separating the assembly of the pre-replication complex (pre-RC) from the initiation of DNA synthesis. A key component of the pre-RC is the hexameric MCM complex, which is also the presumed helicase of the growing forks. An elaborate mechanism recruits the MCM complex to replication origins, and a regulatory chain reaction converts the poised, but inactive, MCM complex into an enzymatically active helicase. A growing list of proteins, including Mcm10 and Cdt1, are involved in the recruitment process. Two protein kinases, the Cdc7-Dbf4 kinase (DDK) and the cyclin-dependent kinase (CDK), trigger a chain reaction that results in the phosphorylation of the MCM complex and finally in the initiation of DNA synthesis. A composite picture from recent studies suggests that DDK is recruited to the pre-RC during G1 phase but must wait until S phase to phosphorylate the MCM complex. CDK is required for the recruitment of Cdc45 and other downstream components of the elongation machinery.

Animals↗

PubMed2003

Budding yeast mcm10/dna43 mutant requires a novel repair pathway for viability.

BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Mcm10p functionally interacts with components of the pre-replicative complex (Mcm2-Mcm7 complex and origin recognition complex) as well as the pre-initiation complex component (Cdc45p) suggesting that it may be a component of the pre-RC as well as the pre-IC. Two-dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Pol delta and Pol epsilon, suggesting that it may play a role in elongation replication. RESULTS: We show that the mcm10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins. We screened for mutations that are lethal in combination with mcm10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with mcm10. These mutants comprised six complementation groups that can be divided into three classes. Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively. Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2. Class 3 includes novel DNA repair genes represented by SLM2 and SLM6. CONCLUSIONS: The viability of the mcm10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks. These results are consistent with the hypothesis that Mcm10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins.

Carrier Proteins↗

PubMed2000

Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins.

MCM2-7, a complex of six subunits, is an essential component of the prereplication chromatin that is assembled at Saccharomyces cerevisiae replication origins during G(1) phase. It is also believed to be the processive helicase at growing forks. To elucidate the action of MCM2-7 during the transition from initiation to elongation replication, we have focused our studies on Mcm10, a replication initiation protein that physically interacts with members of the MCM2-7 complex. We show that Mcm10 is a chromatin-associated protein that mediates the association of the MCM2-7 complex with replication origins. Furthermore, diminished interaction between Mcm10 and Mcm7, a subunit of the MCM2-7 complex, by a mutation in either Mcm10 or Mcm7 inhibits replication initiation. Surprisingly, a double mutant containing both the mcm10-1 and mcm7-1 (cdc47-1) alleles restores interaction between Mcm10 and Mcm7 and corrects all of the defects exhibited by each of the single mutants, including the stalling of replication forks at replication origins typically seen in mcm10-1 cells. This mutual compensation of defects between two independently isolated mutations is allele specific. These results suggest that Mcm10, like Mcm7, is a critical component of the prereplication chromatin and that interaction between Mcm10 and Mcm7 is required for proper replication initiation and prompt release of origin-bound factors.

Alleles↗

PubMed2006

Deregulated minichromosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis.

Minichromosomal maintenance protein 7 (MCM7) is an essential component of the replication helicase complex (MCM2-7) required for DNA replication. Although this function is highly conserved among eukaryotes, additional functions for the MCM molecules continue to be described. Minichromosomal maintenance protein 7 is a marker for proliferation and is upregulated in a variety of tumors including neuroblastoma, prostate, cervical and hypopharyngeal carcinomas. To further investigate the general role of MCM7 in tumorigenesis, we generated a mouse model with deregulated MCM7 expression targeted to the basal layer of the epidermis using the keratin 14 (K14) promoter (K14.MCM7). When subjected to a two-stage chemical carcinogenesis protocol (dimethylbenz[alpha]anthracene (DMBA) initiation with 12-ortho-tetradecanoylphorbol-13-acetate promotion), K14.MCM7 mice showed significantly increased incidence and prevalence of tumor development relative to controls. Furthermore, within 40 weeks of treatment over 45% K14.MCM7 mice exhibited tumors that had converted to squamous cell carcinomas versus none in the control group. As predicted from previous skin carcinogenesis studies using DMBA as the initiating agent, Ras mutations where found in more than 90% of tumors isolated from K14.MCM7 mice. Whereas previous studies have shown that MCM7 is useful as a proliferation marker, our data suggest that deregulated MCM7 expression actively contributes to tumor formation, progression and malignant conversion.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2006

Direct interaction between cohesin complex and DNA replication machinery.

Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase alpha. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.

Cell Cycle Proteins↗

PubMed2000

Cdk2-dependent and -independent pathways in E2F-mediated S phase induction.

The transcription factor E2F plays an important role in G(1) to S phase transition in the higher eukaryotic cell cycle. Although a number of E2F-inducible genes have been identified, the biochemical cascades from E2F to the S phase entry remain to be investigated. In this study, we generated stably transfected mouse NIH3T3 cells that express exogenous human E2F-1 under the control of a heavy metal-inducible metallothionein promoter and analyzed the molecular mechanism of the E2F-1-mediated initiation of chromosomal DNA replication. Ectopic E2F-1 expression in cells arrested in G(0)/G(1) by serum deprivation enabled them to progress through G(1) and to enter S phase. During the G(1) progression, mouse cyclin E, but little of cyclin D1, was induced to express, which subsequently activated Cdk2. Experiments using the Cdk inhibitory proteins p27, p18, and p19 proved that the activity of Cdk2, but not of Cdk4, was required for S phase entry mediated by E2F-1. Minichromosome maintenance proteins (MCM) 4 and 7, the components of the DNA-replication initiation complex (RC), were constitutively expressed during the cell cycle, although the MCM genes are well known E2F-inducible genes. However, tight association of these two proteins with chromatin depended upon ectopic E2F-1 expression. In contrast, the Cdc45 protein, another RC component, which turned out to be a transcriptional target of E2Fs, was induced to express and subsequently bound to chromatin in response to E2F-1. Experiments utilizing a chemical Cdk-specific inhibitor, butyrolactone I, revealed that Cdk2 activity was required only for chromatin binding of the Cdc45 proteins, and not for the expression of Cdc45 or chromatin binding of MCM4 and -7. These results indicate that at least two separate pathways function downstream of E2F to initiate S phase; one depends upon the activity of Cdk2 and the other does not.

3T3 Cells↗

PubMed2005

Pre-replication complex organization in the atypical DNA replication cycle of Plasmodium falciparum: characterization of the mini-chromosome maintenance (MCM) complex formation.

The overall organization of cell division in Plasmodium is unique compared to that observed in model organisms because DNA replicates more than once per cell cycle at several points of its life cycle. The sequencing of the Plasmodium genome has also revealed the apparent absence of many key components (e.g. Cdt1, DDK and Cdc45) of the eukaryotic cell cycle machinery that are responsible for the formation of the pre-replication complex (pre-RC). We have characterized the Plasmodium falciparum minichromosome maintenance complex (MCM) that plays a key role in the transition of pre-RC to the RC. Similar to other eukaryotes, the Plasmodium genome encodes six MCM subunits. Here, we show that expression levels of at least three of the PfMCM subunits, the homologues of MCM2, MCM6 and MCM7, change during the intraerythrocytic development cycle, peaking in schizont and decreasing in the ring and trophozoite stages. PfMCM2, 6 and 7 subunits interact with each other to form a developmentally regulated complex: these interactions are detectable in rings and schizonts, but not in trophozoites. PfMCM2, 6 and 7 subunits are localized in both cytosolic and nucleosolic fractions during all intraerythrocytic stages of P. falciparum development, with increased nuclear localization in schizonts. Only PfMCM6 is associated with the chromatin fraction at all stages of growth. No phosphorylation of PfMCM2, 6 and 7 was detected, but two as yet unidentified threonine-phosphosphorylated proteins were present in the complex, whose pattern of phosphorylation varied during parasite development.

Amino Acid Sequence↗

PubMed1999

Identification and complete cDNA sequence of the missing Drosophila MCMs: DmMCM3, DmMCM6 and DmMCM7.

The minichromosome maintenance (MCM) gene family consists of six members (MCM2, 3, 4, 5, 6 and 7) in Saccharomyces cerevisiae as well as in humans. Each family member plays an essential role in the replication of DNA. In Drosophila melanogaster only three members, DmMCM2, DmMCM4/dpa and DmMCM5/DmCDC46, have been studied. In addition, two other partial sequences were recently reported. Using degenerate primers and low stringency PCR conditions six different DNA sequences were identified with highest sequence similarity to MCM2, 3, 4, 5, 6 and 7. Sequence analysis of full length cDNA clones corresponding to the MCM3, 6 and 7 fragment proves the existence of six MCM genes in Drosophila melanogaster. Strong homology to the human counterparts, mRNA expression analysis and physico-chemical properties suggest a conserved function in DNA replication for DmMCM3, 6 and 7.

Amino Acid Sequence↗

PubMed2000

Inhibition of Mcm4,6,7 helicase activity by phosphorylation with cyclin A/Cdk2.

A strong body of evidence indicates that cyclin-dependent protein kinases are required not only for the initiation of DNA replication but also for preventing over-replication in eukaryotic cells. Mcm proteins are one of the components of the replication licensing system that permits only a single round of DNA replication per cell cycle. It has been reported that Mcm proteins are phosphorylated by the cyclin-dependent kinases in vivo, suggesting that these two factors are cooperatively involved in the regulation of DNA replication. Our group has reported that a 600-kDa Mcm4,6,7 complex has a DNA helicase activity that is probably necessary for the initiation of DNA replication. Here, we examined the in vitro phosphorylation of the Mcm complexes with cyclin A/Cdk2 to understand the interplay between Mcm proteins and cyclin-dependent kinases. The cyclin A/Cdk2 mainly phosphorylated the amino-terminal region of Mcm4 in the Mcm4,6,7 complex. The phosphorylation was associated with the inactivation of its DNA helicase activity. These results raise the possibility that the inactivation of Mcm4,6,7 helicase activity by Cdk2 is a part of the system for regulating DNA replication.

Animals↗

PubMed2000

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.

Animals↗

PubMed2001

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.

Cell Cycle Proteins↗

PubMed2002

Two mcm3 mutations affect different steps in the initiation of DNA replication.

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.

Alleles↗

PubMed1999

Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity.

Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2, 4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.

Adenosine Triphosphatases↗

PubMed1999

Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase alpha.

Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase. We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively. A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610-amino acid protein was comprised of 20 exons. One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells. Splicing was different in HeLa cells and TIG-1 cells, a human diploid cell line. Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase alpha. The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase alpha on to the DNA replication complex through binding to hMCM7.

Alternative Splicing↗

PubMed1998

Human papillomavirus oncoprotein E6 binds to the C-terminal region of human minichromosome maintenance 7 protein.

Oncoprotein E6 of the human papillomavirus (HPV) associated with cervical cancer (HPV-16 and -18) degrades tumor suppressor protein p53, but seems to have p53-independent transforming functions. We searched for other cellular targets for the N-terminal region of HPV-16 E6 using a yeast two-hybrid system. The E6 was found to bind to the C-terminal region of a human minichromosome maintenance 7 (hMCM7) protein, which is a component of replication licensing factors. The full-length hMCM7 translated in vitro was capable of binding to bacterially expressed E6. In yeast cells the E6s of the cancer-associated HPVs (HPV-16, -18, and -58) bound to hMCM7 more strongly than those of the HPVs associated with a benign tumor (HPV-6 and -11). Binding of E6 with hMCM7 may cause chromosomal abnormalities found in the human cells expressing E6s of oncogenic HPVs.

Binding Sites↗

PubMed2000

The MAT1 cyclin-dependent kinase-activating kinase (CAK) assembly/targeting factor interacts physically with the MCM7 DNA licensing factor.

MAT1 (ménage à trois1) functions as an assembly/targeting factor of CAK (cyclin-dependent kinase-activating kinase). In a search for MAT1-interacting proteins using yeast two-hybrid system, MCM7 (minichromosome maintenance 7), a member of a family of DNA licensing factors, was identified. The physical interaction between MAT1 and MCM7 was confirmed in vivo in yeast cells and verified with in vitro protein binding assays. Further studies showed the RING-finger motif of MAT1 is not required for the interaction with MCM7, while the C-terminal domain of MAT1 is indispensable. Immunoprecipitation of MCM7 in human osteosarcoma MG63 cells demonstrated that MCM7 associates with the CAK complex in vivo.

Amino Acid Motifs↗

PubMed2003

Comparative analysis of cervical cancer in women and in a human papillomavirus-transgenic mouse model: identification of minichromosome maintenance protein 7 as an informative biomarker for human cervical cancer.

Human papillomaviruses (HPVs), such as HPV-16, are associated with >99% of cervical cancers in women. Two viral oncogenes, E6 and E7, are selectively expressed in these cancers. K14E6 and K14E7 transgenic mouse strains, which express the HPV16 E6 or E7 gene in stratified squamous epithelia, display many acute and long-term phenotypes indicative of the oncogenic potential of E6 and E7 including epithelial hyperplasia, abrogation of normal DNA damage responses, and spontaneous skin tumors. When treated with estrogen, these HPV-16 transgenic mice develop a progressive disease leading to cervical cancer that shows many histopathological parallels to cervical cancer in women. In this study, we evaluated the cervical lesions that arise in these transgenic mice for the expression of biomarkers induced in human cervical cancer. These analyses, which showed close parallels in the timing and pattern of expression of cyclin E and Ki-67 in the mouse cervical disease compared with that in humans, provided further validation of this HPV-16 transgenic mouse model for human cervical cancer. We then used our mouse model to identify minichromosome maintenance protein 7 (MCM7), an E2F-induced cellular DNA replication factor, as a novel biomarker for cervical cancer. In both the mouse and human disease, strong, full thickness staining for MCM7 was seen selectively in the epithelium of high-grade intraepithelial lesions and in frank cancer. The uniform staining pattern and strong signal for MCM7 suggest that MCM7 may be a highly informative biomarker for cervical cancer.

Animals↗