Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex.

In eukaryotes, tight regulatory mechanisms ensure the ordered progression through the cell cycle phases. The mechanisms that prevent chromosomal DNA replication from taking place more than once each cell cycle are thought to involve the function of proteins of the minichromosome maintenance (MCM) family. Here, we demonstrate that Xenopus MCM4, a member of the MCM protein family related to Spcdc21/ ScCDC54, is part of a large protein complex comprising several other MCM proteins. MCM4 undergoes cell cycle-dependent phosphorylation both in cleaving embryos and in cell-free extracts. MCM4 phosphorylation starts concomitantly with the clearing of the MCM complex from the chromatin during S phase. Phosphorylation is carried out by cdc2/cyclinB protein kinase, which phosphorylates MCM4 in vitro at identical sites as the ones phosphorylated in vivo. Phosphorylation is specific for cdc2 protein kinase since MCM4 is not a substrate for other members of the cdk family. Furthermore, phosphorylation of MCM4 dramatically reduces its affinity for the chromatin. We propose that the cell cycle-dependent phosphorylation of MCM4 is a mechanism which inactivates the MCM complex from late S phase through mitosis, thus preventing illegitimate DNA replication during that period of the cell cycle.

Prognostic significance of minichromosome maintenance proteins in breast cancer

A role for the minichromosome maintenance (MCM) proteins in cancer initiation and progression is slowly emerging. Functioning as a complex to ensure a single chromosomal replication per cell cycle, the six family members have been implicated in several neoplastic disease states, including breast cancer. Our study aim to investigate the prognostic significance of these proteins in breast cancer. We studied the expression of MCMs in various datasets and the associations of the expression with clinicopathological parameters. When considered alone, high level MCM4 overexpression was only weakly associated with shorter survival in the combined breast cancer patient cohort (n = 1441, Hazard Ratio = 1.31; 95% Confidence Interval = 1.11-1.55; p = 0.001). On the other hand, when we studied all six components of the MCM complex, we found that overexpression of all MCMs was strongly associated with shorter survival in the same cohort (n = 1441, Hazard Ratio = 1.75; 95% Confidence Interval = 1.31-2.34; p <0.001), suggesting these MCM proteins may cooperate to promote breast cancer progression. Indeed, their expressions were significantly correlated with each other in these cohorts. In addition, we found that increasing number of overexpressed MCMs was associated with negative ER status as well as treatment response. Together, our findings are reproducible in seven independent breast cancer cohorts, with 1441 patients, and suggest that MCM profiling could potentially be used to predict response to treatment and prognosis in breast cancer patients.

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.

The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ΔH contains DNA helicase activity

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508–24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum ΔH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric (≈103 kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3′-to-5′ ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.

A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase

The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. Here we have expressed and purified the M. thermoautotrophicum MCM protein. The purified protein forms a complex that has a molecular mass of ≈850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3′ to 5′ helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase. It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea. The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.

Nuclear Localization of Schizosaccharomyces pombe Mcm2/Cdc19p Requires MCM Complex Assembly

The minichromosome maintenance (MCM) proteins MCM2–MCM7 are conserved eukaryotic replication factors that assemble in a heterohexameric complex. In fission yeast, these proteins are nuclear throughout the cell cycle. In studying the mechanism that regulates assembly of the MCM complex, we analyzed the cis and trans elements required for nuclear localization of a single subunit, Mcm2p. Mutation of any single mcm gene leads to redistribution of wild-type MCM subunits to the cytoplasm, and this redistribution depends on an active nuclear export system. We identified the nuclear localization signal sequences of Mcm2p and showed that these are required for nuclear targeting of other MCM subunits. In turn, Mcm2p must associate with other MCM proteins for its proper localization; nuclear localization of MCM proteins thus requires assembly of MCM proteins in a complex. We suggest that coupling complex assembly to nuclear targeting and retention ensures that only intact heterohexameric MCM complexes remain nuclear.

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.

The future for genetic studies in reproduction

Genetic factors contribute to risk of many common diseases affecting reproduction and fertility. In recent years, methods for genome-wide association studies(GWAS) have revolutionized gene discovery forcommontraits and diseases. Results of GWAS are documented in the Catalog of Published Genome-Wide Association Studies at the National Human Genome Research Institute and report over 70 publications for 32 traits and diseases associated with reproduction. These include endometriosis, uterine fibroids, age at menarche and age at menopause. Results that pass appropriate stringent levels of significance are generally well replicated in independent studies. Examples of genetic variation affecting twinning rate, infertility, endometriosis and age at menarche demonstrate that the spectrum of disease-related variants for reproductive traits is similar to most other common diseases.GWAS 'hits' provide novel insights into biological pathways and the translational value of these studies lies in discovery of novel gene targets for biomarkers, drug development and greater understanding of environmental factors contributing to disease risk. Results also show that genetic data can help define sub-types of disease and co-morbidity with other traits and diseases. To date, many studies on reproductive traits have used relatively small samples. Future genetic marker studies in large samples with detailed phenotypic and clinical information will yield new insights into disease risk, disease classification and co-morbidity for many diseases associated with reproduction and infertility.

Whole blood transcriptional profiling in ankylosing spondylitis identifies novel candidate genes that might contribute to the inflammatory and tissue-destructive disease aspects

Introduction: A number of genetic-association studies have identified genes contributing to ankylosing spondylitis (AS) susceptibility but such approaches provide little information as to the gene activity changes occurring during the disease process. Transcriptional profiling generates a 'snapshot' of the sampled cells' activity and thus can provide insights into the molecular processes driving the disease process. We undertook a whole-genome microarray approach to identify candidate genes associated with AS and validated these gene-expression changes in a larger sample cohort. Methods: A total of 18 active AS patients, classified according to the New York criteria, and 18 gender- and age-matched controls were profiled using Illumina HT-12 whole-genome expression BeadChips which carry cDNAs for 48,000 genes and transcripts. Class comparison analysis identified a number of differentially expressed candidate genes. These candidate genes were then validated in a larger cohort using qPCR-based TaqMan low density arrays (TLDAs). Results: A total of 239 probes corresponding to 221 genes were identified as being significantly different between patients and controls with a P-value <0.0005 (80% confidence level of false discovery rate). Forty-seven genes were then selected for validation studies, using the TLDAs. Thirteen of these genes were validated in the second patient cohort with 12 downregulated 1.3- to 2-fold and only 1 upregulated (1.6-fold). Among a number of identified genes with well-documented inflammatory roles we also validated genes that might be of great interest to the understanding of AS progression such as SPOCK2 (osteonectin) and EP300, which modulate cartilage and bone metabolism. Conclusions: We have validated a gene expression signature for AS from whole blood and identified strong candidate genes that may play roles in both the inflammatory and joint destruction aspects of the disease.

The prognostic significance of protein tyrosine phosphatase 4A2 in breast cancer

Although PTP4A3 has been shown to be a very important factor in promoting cancer progression, the role of its close family member PTP4A2 is still largely unknown. Recent reports have shown contradicting results on the role of PTP4A2 in breast cancer progression. Considering this, we aimed to investigate the prognostic value of PTP4A2 in five independent breast cancer data sets (minimum 198 patients per cohort, totaling 1,124 patients) in the Gene Expression Omnibus Database. We found that high expression of PTP4A2 was a favorable prognostic marker in all five independent breast cancer data sets, as well as in the combined cohort, with a hazard ratio of 0.68 (95% confidence interval =0.56-0.83; P<0.001). Low PTP4A2 expression was associated with estrogen receptor-negative tumors and tumors with higher histological grading; furthermore, low expression was inversely correlated with the expression of genes involved in proliferation, including MKI67 and the MCM gene family encoding the minichromosome maintenance proteins. These findings suggest that PTP4A2 may play a role in breast cancer progression by dysregulating cell proliferation. PTP4A2 expression was positively correlated with ESR1, the gene encoding estrogen receptor-alpha, and inversely correlated with EGFR expression, suggesting that PTP4A2 may be involved in these two important oncogenic pathways. Together, our results suggest that expression of PTP4A2 is a favorable prognostic marker in breast cancer.

Cellular Proliferation in the Prognosis of Intermediate Grade Mast Cell Tumour in Dogs

This was a retrospective study including ninety samples of dogs with a histological diagnosis of intermediate grade cutaneous mast cell tumour (MCT). The objectives of the study were to validate Minichromosome Maintenance Protein 7 (MCM7) as a prognostic marker in MCTs and to compare the ability of mitotic index (MI), Ki67 and MCM7 to predict outcome. The median survival for the entire population was not reached at 2099 days. The mean survival time was 1708 days. Seventy-two cases were censored after a median follow up of 1136 days and eighteen dogs died for causes related to the MCT after a median of 116 days. For each sample MI, Ki67 and MCM7 were determined. The Receiver Operating Characteristic (ROC) curve was obtained for each prognostic marker to evaluate the performance of the test, expressed as area under the curve, and whether the published threshold value was adequate. Kaplan-Meier and corresponding logrank test for MI, Ki67 and MCM7 as binary variables was highly significant (P<0.0001). Multivariable regression analysis of MI, Ki67 and MCM7 corrected for age and surgical margins indicated that the higher risk of dying of MCT was associated with MCM7 > 0.18 (Hazard Ration [HR] 14.7; P<0.001) followed by MI > 5 (HR 13.9; P<0.001) and Ki67 > 0.018 (HR 8.9; P<0.001). Concluding, the present study confirmed that MCM7 is an excellent prognostic marker in cutaneous MCTs being able to divide Patnaik intermediate grade tumours in two categories with different prognosis. Ki67 was equally good confirming its value as a prognostic marker in intermediate grade MCTs. The mitotic index was extremely specific, but lacked of sensitivity. Interestingly, mitotic index, Ki67 and MCM7 were independent from each other suggesting that their combination would improve their individual prognostic value.

Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-γ

In response to IFN-γ, the latent cytoplasmic Stat1 (signal transducer and activator of transcription) proteins translocate into the nucleus and activate transcription. We showed previously that Stat1 recruits a group of nuclear proteins, among them MCM5 (minichromosome maintenance) and MCM3, for transcription activation. MCM5 directly interacts with the transcription activation domain (TAD) of Stat1 and enhances Stat1-mediated transcription activation. In this report, we identified two specific residues (R732, K734) in MCM5 that are required for the direct interaction between Stat1 and MCM5 both in vitro and in vivo. MCM5 containing mutations of R732/K734 did not enhance Stat1-mediated transcription activation in response to IFN-γ. In addition, it also failed to form complexes with other MCM proteins in vivo, suggesting that these two residues may be important for an interaction domain in MCM5. Furthermore, MCM5 bearing mutations in its ATPase and helicase domains did not enhance Stat1 activity. In vitro binding assays indicate that MCM3 does not interact directly with Stat1, suggesting that the presence of MCM3 in the group of Stat1TAD-interacting proteins is due to the association of MCM3 with MCM5. Finally, gel filtration analyses of nuclear extracts from INF-γ-treated cells demonstrate that there is a MCM5/3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5/3 subcomplex through direct interaction with MCM5 in the process of IFN-γ-induced gene activation.

Implication des protéines MCM dans la réponse cellulaire aux dommages à l’ADN

Les protéines MCM (minichromosome maintenance) forment un complexe hétérohexamérique composé des protéines MCM2 à MCM7 qui possède une activité hélicase nécessaire lors de la réplication de l’ADN. Ce complexe est la cible des protéines ATM et ATR, kinases responsables de l’initiation de la réponse cellulaires aux dommages à l’ADN, pour permettre l’arrêt de la réplication lors de la détection de cassure double brin. De plus, les MCM permettent le remodelage de la chromatine par leur activité hélicase mais aussi par leur association avec une chaperone d’histone la protéine ASF1. Toutefois, la majorité des complexes MCM ne co-localisent pas avec les origines de réplication. De plus, la quantité des protéines MCM dans la cellule est nettement supérieure à la quantité requise lors de la réplication. Ces deux faits laissent présager que ce complexe hélicase pourrait jouer un second rôle. Des études effectuées au laboratoire ont démontré une augmentation de la fixation à la chromatine des protéines MCM suite au traitement avec l’étoposide, un inhibiteur de la topoisomérase II qui cause des cassures double brin. L’étude des interactions de la protéine MCM2 par spectrométrie de masse ainsi que par immunobuvardage ont démontré une augmentation de l’interaction entre la protéine MCM2 et ASF1 suite aux dommages. Ceci suggère que les protéines MCM pourraient être impliquées dans les mécanismes de réparation de l’ADN. La nature de l’interaction entre la protéine MCM2 et ASF1 a été déterminée in vitro par des immunobuvardages de type Far western et des Dot blot avec des mutants de la protéine MCM2. Des cellules U2OS-Flp-in ont été utilisées pour générer des lignées stables exprimants les protéines MCM2 à MCM7 avec une étiquette GFP ou fusionnées avec une biotine-ligase (BirA). Les cellules ont été cultivées dans du milieu SILAC et des immunoprécipitations ont été effectuées sur des cellules contrôles (R0K0), des cellules qui expriment MCM-GFP ou BirA (R6K4) non-traitées et des cellules qui expriment MCM-GFP ou BirA traitées à l’étoposide (R10K8). Les immunoprécipitations ont été analysés au spectromètre de masse pour déterminer la modulation des interactions avant et après dommages à l’ADN. Les études d’interactions in vitro ont permis d’identifier que l’interaction entre la protéine MCM2 et ASF1 se situe entre les acides aminés 81-162 sur la protéine MCM2. L’approche de spectrométrie de masse a permis d’identifier plusieurs protéines liant le complexe MCM qui sont impliquées non seulement dans la réplication de l’ADN mais aussi dans le remodelage de la chromatine. De plus, certains de ces nouveaux partenaires augmentent leur interaction avec le complexe suite à l’induction de dommages. Ces résultats suggèrent que les protéines MCM jouent un rôle dans la réorganisation de la chromatine dans les mécanismes de réparation de l’ADN.

Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.