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Results for “Minichromosome Maintenance Complex Component 6”

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PubMed1996

A novel human Mcm protein: homology to the yeast replication protein Mis5 and chromosomal location.

Mcm proteins perform functions related to the regulation of eukaryotic genome replication. Previous work has shown that human cells contain at least five different Mcm proteins. We report now the amino acid sequence of an additional human Mcm protein, p105Mcm, and show that it is homologous to the Schizosaccharomyces pombe protein Mis5. We demonstrate that the gene for protein p105Mcm (HGMW-approved symbol, MCM6) is located on human chromosome 2q14-q21. All six known human Mcm proteins have now been sequenced and compared to their yeast counterparts.

Amino Acid Sequence↗
PubMed1996

Characterisation of a human homologue of a yeast cell division cycle gene, MCM6, located adjacent to the 5' end of the lactase gene on chromosome 2q21.

Four exons of a human homologue of a yeast cell division cycle gene (MCM6/mis5, which is thought to encode a DNA replication licensing factor) have been identified 3.3 kb upstream from the start of transcription of the intestinal lactase gene on human chromosome 2q21, initially by similarity to a rat 'intestinal crypt-cell replication factor'. RT-PCR analysis shows, that unlike lactase, MCM6 is not restricted in its tissue distribution and does not show person-to-person variation in the level of expression in adult intestine.

Adult↗
PubMed1999

Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F.

Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters.

Animals↗
PubMed2004

Polyomavirus tumorantigens have a profound effect on gene expression in mouse fibroblasts.

Polyomavirus (Py) large and small tumorantigens together are competent to induce S phase in growth-arrested mouse fibroblasts. The capacity of the large tumorantigen to bind the pocket proteins, pRB, p130 and p107, is important for the transactivation of DNA synthesis enzymes and the cyclins E and A, while the interference of small tumorantigen with protein phosphatase PP2A causes a destabilization of the cdk2 inhibitor p27, and thus leads to strong cyclin E- and cyclin A-dependent cdk2 activity. Py small tumorantigen, in addition, is able to transactivate cyclin A. Hence, this protein might have a much wider effect on gene expression in arrested mouse fibroblasts than hitherto suspected. This may have a profound part in the known capacity of Py to form tumors in mice. Therefore, it was interesting to gain an insight into the spectrum of transcriptional deregulation by Py tumorantigens. Accordingly, we performed microarray analysis of quiescent mouse fibroblasts in the absence and presence of small or large tumorantigen. We found that the viral proteins can induce or repress a great variety of genes beyond those involved in the S phase induction and DNA synthesis. The results of the microarray analysis were confirmed for selected genes by several methods, including real-time PCR. Interestingly, a mutation of the binding site for pocket proteins in case of LT and for PP2A in case of ST has a variable effect on the deregulation of genes by the viral proteins depending on the gene in question. In fact, some genes are transactivated by LT as well as ST completely independent of an interaction with their major cellular targets, pocket proteins and PP2A, respectively.

3T3 Cells↗
PubMed1996

Mouse MCM proteins: complex formation and transportation to the nucleus.

BACKGROUND: The members of the MCM protein family, including MCM2, MCM3, Cdc21, CDC46, Mis5 and CDC47, are considered to be involved in the control of a single round of DNA replication during S phase in eukaryotes. They bind to chromatin during G1 and detach from it during S phase as if they license the chromatin to replicate. However, unlike the originally proposed 'licensing factor' and the budding yeast homologues, mammalian MCM2 and P1MCM3 proteins appeared to be localized in the nucleus during the interphase. RESULTS: We purified mCdc21 and its associated proteins from mouse cell extract by anti-mCdc21 immunoaffinity chromatography. Three proteins which co-purified with mCdc21 were identified as mCDC47, mMis5 and mMCM2, all were MCM proteins. Glycerol gradient centrifugation analysis showed that all the mouse MCM proteins were detected at 450-600 kDa, an indication of the sum of their calculated molecular weights from their amino acid sequences. mCdc21 was displaced from replicated chromatin in a similar way to P1MCM3 and MCM2 during S phase. Among the six mouse MCM proteins, only mMCM2 and mP1MCM3 showed nuclear localization when overexpressed in COS cells. CONCLUSIONS: We conclude that in the mouse, six MCM proteins form a single protein complex of molecular weight 450-600 kDa, which may enter the nucleus by nuclear localization signals in the mMCM2 and mP1MCM3 subunits.

Amino Acid Sequence↗
PubMed1997

HsMCM6: a new member of the human MCM/P1 family encodes a protein homologous to fission yeast Mis5.

BACKGROUND: The tight regulatory mechanism that prevents more than one round of chromosomal DNA replication per cell cycle is thought to require the function of Mcm/P1 proteins. We report here the structural and functional analyses of HsMcm6, a human homologue of the Mis5 of Schizosaccharomyces pombe. RESULTS: We demonstrate here that the transcription of the HsMCM6 gene was repressed in quiescent cells but was rapidly induced at the G1/S phase by growth factor stimulation. The 5' regulatory region of the HsMCM6 gene was found to harbour four putative E2F binding motifs, and these were responsible for the promoter activity. The HsMcm6 protein level oscillated during the cell cycle, with a peak at the G1/S phase. We also showed that the cell-cycle dependent change of subcellular localization of HsMcm6 resembles those of other Mcm/P1 proteins. HsMcm6 consists of two forms, a form extractable by Nonidet P-40 and the nucleus-bound form. A demonstration of the association of HsMcm6 with HsMcm2 and HsMcm7 in vivo supports the idea that they behave as a heteromeric complex. We mapped the HsMCM6 gene at 2q12-14. CONCLUSION: The results indicate that the behaviour of HsMcm6 is reminiscent of replication licensing factor like other Mcm/P1 family members.

Amino Acid Sequence↗
PubMed1998

Human protein MCM6 on HeLa cell chromatin.

Minichromosome maintenance (Mcm) proteins perform essential functions regulating the replication of chromatin. Human cells, like other eukaryotic cells, express at least six Mcm proteins conserved in the central region. We have earlier described the primary structures of five human Mcm proteins, but the primary structure of the sixth human Mcm protein, MCM6, was identified only recently. We now use antibodies, specific for the MCM6 protein, to assess its intranuclear distribution. We find that a fraction of MCM6 protein occurs in the nucleosol, forming multiprotein complexes with other Mcm proteins. More importantly, we use for the first time micrococcal nuclease as a tool to investigate the association of MCM6 protein with chromatin. After short digestion times, a considerable fraction of the MCM6 protein is released from chromatin as a multiprotein complex that includes other Mcm proteins as well. In addition, fractions of MCM3 and MCM6 proteins are released by nuclease digestion as monomeric proteins indicating that at least these two Mcm proteins may also occur as single molecules on chromatin. The data also suggest that the chromatin regions with bound Mcm proteins are more vulnerable to nuclease attack than bulk chromatin and may therefore differ in the arrangement of nucleosomes.

Antibodies↗
PubMed2005

Caenorhabditis elegans geminin homologue participates in cell cycle regulation and germ line development.

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins.

Amino Acid Sequence↗
PubMed2002

Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity.

DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.

Amino Acid Sequence↗
PubMed2006

The replicative helicases of bacteria, archaea, and eukarya can unwind RNA-DNA hybrid substrates.

Replicative helicases are hexameric enzymes that unwind DNA during chromosomal replication. They use energy from nucleoside triphosphate hydrolysis to translocate along one strand of the duplex DNA and displace the complementary strand. Here, the ability of a replicative helicase from each of the three domains, bacteria, archaea, and eukarya, to unwind RNA-containing substrate was determined. It is shown that all three helicases can unwind DNA-RNA hybrids while translocating along the single-stranded DNA. No unwinding could be observed when the helicases were provided with a single-stranded RNA overhang. Using DNA, RNA, and DNA-RNA chimeric oligonucleotides it was found that whereas the enzymes can bind both DNA and RNA, they could translocate only along DNA and only DNA stimulates the ATPase activity of the enzymes. Recent observations suggest that helicases may interact with enzymes participating in RNA metabolism and that RNA-DNA hybrids may be present on the chromosomes. Thus, the results presented here may suggest a new role for the replicative helicases during chromosomal replication or in other cellular processes.

Adenosine Triphosphatases↗
PubMed2004

A novel method for diagnosis of adult hypolactasia by genotyping of the -13910 C/T polymorphism with Pyrosequencing technology.

The lactose-loading test and other functional tests that have been the most widely used clinically in the diagnosis of adult hypolactasia up to now are labour intensive and costly, and suffer from low sensitivity as well as low specificity. In addition, lactose-loading tests may be painful to the patient. Here, a new genotyping method for the diagnosis of adult hypolactasia is described. The method utilizes Pyrosequencing technology, which gives the DNA sequence around the recently identified C/T polymorphic site in the MCM6 gene. Among the advantages compared to the other genotyping methods published are less staff hands-on time than for example RFLP analyses, and the avoidance of radioactivity, as in the originally described isotope-minisequencing. Most importantly, Pyrosequencing, which is a direct DNA sequencing technique, gives unambiguous genotyping results as well as some redundant sequence information beyond the SNP position, which serves as a valuable internal control, obtained for each sample.

Adult↗
PubMed2002

SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast.

Initiation of DNA replication in eukaryotic cells is regulated through the ordered assembly of replication complexes at origins of replication. Association of Cdc45 with the origins is a crucial step in assembly of the replication machinery, hence can be considered a target for the regulation of origin activation. To examine the process required for SpCdc45 loading, we isolated fission yeast SpSld3, a counterpart of budding yeast Sld3 that interacts with Cdc45. SpSld3 associates with the replication origin during G1-S phases and this association depends on Dbf4-dependent (DDK) kinase activity. In the corresponding period, SpSld3 interacts with minichromosome maintenance (MCM) proteins and then with SpCdc45. A temperature-sensitive sld3-10 mutation suppressed by the multicopy of the sna41+ encoding SpCdc45 impairs loading of SpCdc45 onto chromatin. In addition, this mutation leads to dissociation of preloaded Cdc45 from chromatin in the hydroxyurea-arrested S phase, and DNA replication upon removal of hydroxyurea is retarded. Thus, we conclude that SpSld3 is required for stable association of Cdc45 with chromatin both in initiation and elongation of DNA replication. The DDK-dependent origin association suggests that SpSld3 is involved in temporal regulation of origin firing.

Amino Acid Sequence↗
PubMed1994

Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality.

Precise chromosome transmission in cell division cycle is maintained by a number of genes. The attempt made in the present study was to isolate temperature-sensitive (ts) fission yeast mutants that display high loss rates of minichromosomes at permissive or semipermissive temperature (designated mis). By colony color assay of 539 ts strains that contain a minichromosome, we have identified 12 genetic loci (mis1-mis12) and determined their phenotypes at restrictive temperature. Seven of them are related to cell cycle block phenotype at restrictive temperature, three of them in mitosis. Unequal distribution of regular chromosomes in the daughters is extensive in mis6 and mis12. Cells become inviable after rounds of cell division due to missegregation. The phenotype of mis5 is DNA replication defect and hypersensitivity to UV ray and hydroxyurea. mis5+ encodes a novel member of the ubiquitous MCM family required for the onset of replication. The mis5+ gene is essential for viability and functionally distinct from other previously identified members in fission yeast, cdc21+, nda1+, and nda4+. The mis11 mutant phenotype was the cell division block with reduced cell size. Progression of the G1 and G2 phases is blocked in mis11. The cloned mis11+ gene is identical to prp2+, which is essential for RNA splicing and similar to a mammalian splicing factor U2AF65.

Amino Acid Sequence↗
PubMed2004

A novel intermediate in initiation complex assembly for fission yeast DNA replication.

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.

Cell Cycle↗
PubMed1999

Rereplication phenomenon in fission yeast requires MCM proteins and other S phase genes.

The fission yeast Schizosaccharomyces pombe can be induced to perform multiple rounds of DNA replication without intervening mitoses by manipulating the activity of the cyclin-dependent kinase p34(cdc2). We have examined the role in this abnormal rereplication of a large panel of genes known to be involved in normal S phase. The genes analyzed can be grouped into four classes: (1) those that have no effect on rereplication, (2) others that delay DNA accumulation, (3) several that allow a gradual increase in DNA content but not in genome equivalents, and finally, (4) mutations that completely block rereplication. The rereplication induced by overexpression of the CDK inhibitor Rum1p or depletion of the Cdc13p cyclin is essentially the same and requires the activity of two minor B-type cyclins, cig1(+) and cig2(+). In particular, the level, composition, and localization of the MCM protein complex does not alter during rereplication. Thus rereplication in fission yeast mimics the DNA synthesis of normal S phase, and the inability to rereplicate provides an excellent assay for novel S-phase mutants.

Cell Cycle↗
PubMed2006

Gene expression profiling of primary cutaneous melanoma and clinical outcome.

BACKGROUND: Gene expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors. To identify differentially expressed genes that may be involved in melanoma progression and prognosis, we investigated the relationship between gene expression profiles and clinical outcome in a cohort of patients with primary melanoma. METHODS: Labeled complementary RNA (cRNA) from each tissue sample was hybridized to a pangenomic 44K 60-mer oligonucleotide microarray. Class comparison and class prediction analyses were performed to identify genes whose expression in primary melanomas was associated with 4-year distant metastasis-free survival among 58 patients with at least 4 years of follow-up, distant metastasis, or death. Results were validated immunohistochemically at the protein level in 176 independent primary melanomas from patients with a median clinical follow-up of 8.5 years. Survival was analyzed with a Cox multivariable model and stratified log-rank test. All statistical tests were two-sided. RESULTS: We identified 254 genes that were associated with distant metastasis-free survival of patients with primary melanoma. These 254 genes include genes involved in activating DNA replication origins, such as minichromosome maintenance genes and geminin. Twenty-three of these genes were studied at the protein level; expression of five (MCM4, P = .002; MCM3, P = .030; MCM6, P = .004; KPNA2, P = .021; and geminin, P = .004) was statistically significantly associated with overall survival in the validation set. In a multivariable Cox model adjusted for tumor thickness, ulceration, age, and sex, expression of MCM4 (hazard ratio [HR] of death = 4.04, 95% confidence interval [CI] = 1.39 to 11.76; P = .010) and MCM6 (HR of death = 7.42, 95% CI = 1.99 to 27.64; P = .003) proteins was still statistically significantly associated with overall survival. CONCLUSION: We identified 254 genes whose expression was associated with metastatic dissemination of cutaneous melanomas. These genes may shed light on the molecular mechanisms underlying poor prognosis in melanoma patients.

Adolescent↗
PubMed2004

Self-assembling protein microarrays.

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

Cell Cycle Proteins↗