MCM4 acts as a biomarker for LUAD prognosis

MCM4 forms the pre-replication complex (MCM2-7) with five other minichromosome maintenance (MCM) proteins. This complex binds to replication origins at G1 stage in cell cycle process, playing a critical role in DNA replication initiation. Recently, MCM4 is reported to have a complex interaction with multiple cancer progression, including gastric, ovarian and cervical cancer. Here, this study mainly focused on the expression of MCM4 and its values in lung adenocarcinoma (LUAD). MCM4 was highly expressed in LUAD tumours and cells, and had an important effect on the overall survival. Overexpression of MCM4 promoted the proliferation, and suppressed the apoptosis in LUAD cells. However, MCM4 silence led to the opposite results. In vivo, knockdown of MCM4 inhibited tumour volume and weight in xenograft mouse model. As a member of DNA helicase, knockdown of MCM4 caused cell cycle arrest at G1 stage through inducing the expression of P21, a CDK inhibitor. These findings indicate that MCM4 may be a possible new therapeutic target for LUAD in the future.     

微小染色体维持蛋白与肿瘤

微小染色体维持(minichromosome maintenance,MCM)蛋白质家族是DNA复制前复合物的重要组成部分,在DNA复制启动过程和DNA损伤修复中发挥着重要作用.MCM蛋白质家族成员在转录调节、染色质重塑和检查点应答中也扮演着重要角色.最近研究发现,MCM异常可导致恶性肿瘤的发生发展,在不同肿瘤(前列腺癌、子宫内膜癌、卵巢癌、肝癌、肺癌、胶质母细胞瘤和髓母细胞瘤等)中呈现异常表达并与肿瘤细胞增殖、侵袭、转移能力密切相关,MCM蛋白有望作为临床上诊断相关恶性肿瘤及提示其预后的生物学标志物.更为重要的是,MCM蛋白质复合物晶体结构逐步得到解析,这不仅有利于阐明其生理和病理作用与调控机制,亦有助于发现靶向MCM的特异性小分子抑制剂,为新型抗肿瘤药物的研发提供新思路.     

Plant MCM proteins: role in DNA replication and beyond

Mini-chromosome maintenance (MCM) proteins form heterohexameric complex (MCM2–7) to serve as licensing factor for DNA replication to make sure that genomic DNA is replicated completely and accurately once during S phase in a single cell cycle. MCMs were initially identified in yeast for their role in plasmid replication or cell cycle progression. Each of six MCM contains highly conserved sequence called “MCM box”, which contains two ATPase consensus Walker A and Walker B motifs. Studies on MCM proteins showed that (a) the replication origins are licensed by stable binding of MCM2–7 to form pre-RC (pre-replicative complex) during G1 phase of the cell cycle, (b) the activation of MCM proteins by CDKs (cyclin-dependent kinases) and DDKs (Dbf4-dependent kinases) and their helicase activity are important for pre-RC to initiate the DNA replication, and (c) the release of MCMs from chromatin renders the origins “unlicensed”. DNA replication licensing in plant is, in general, less characterized. The MCMs have been reported from Arabidopsis, maize, tobacco, pea and rice, where they are found to be highly expressed in dividing tissues such as shoot apex and root tips, localized in nucleus and cytosol and play important role in DNA replication, megagametophyte and embryo development. The identification of six MCM coding genes from pea and Arabidopsis suggest six distinct classes of MCM protein in higher plant, and the conserved function right across the eukaryotes. This overview of MCMs contains an emphasis on MCMs from plants and the novel role of MCM6 in abiotic stress tolerance.     

Multiomics profiling of the expression and prognosis of MCMs in endometrial carcinoma

Minichromosome maintenance (MCM) family members are a group of genes involved in regulating DNA replication and cell division and have been identified as oncogenes in various cancer types. Several experimental studies have suggested that MCMs are dysregulated in endometrial carcinoma (EC). However, the expression pattern, clinical value and functions of different MCMs have yet to be analyzed systematically and comprehensively. We analyzed expression, survival rate, DNA alteration, PPT network, GGI network, functional enrichment cancer hallmarks and drug sensitivity of MCMs in patients with EC based on diverse datasets, including Oncomine, GEPIA, Kaplan–Meier Plotter, HPA, Sangerbox and GSCALite databases. The results indicated that most MCM members were increased in EC and showed a prognostic value in survival analysis, which were considerately well in terms of PFS and OS prognostic prediction. Importantly, functional enrichment, PPI network and GGI network suggested that MCMs interact with proteins related to DNA replication and cell division, which may be the mechanism of MCM promote EC progression. Further data mining illustrated that MCMs have broad DNA hypomethylation levels and high levels of copy number aberrations in tumor tissue samples, which may be the mechanism causing the high expression level of MCMs. Moreover, MCM2 can activate or suppress diverse cancer-related pathways and is implicated in EC drug sensitivity. Taking together, our findings illustrate the expression pattern, clinical value and function of MCMs in EC and imply that MCMs are potential targets for precision therapy and new biomarkers for the prognosis of patients with EC.     

Interaction of heliquinomycin with single-stranded DNA inhibits MCM4/6/7 helicase

The antibiotic heliquinomycin inhibited cellular DNA replication at IC(50) of 2.5 μM without affecting level of chromatin-bound MCM4 and without activating the DNA replication stress checkpoint system, suggesting that heliquinomycin perturbs DNA replication mainly by inhibiting the activity of replicative DNA helicase that unwinds DNA duplex at replication forks. Among the DNA helicases involved in DNA replication, DNA helicase B was inhibited by heliquinomycin at IC(50) of 4.3 μM and RECQL4 helicase at IC(50) of 14 μM; these values are higher than that of MCM4/6/7 helicase (2.5 μM). These results suggest that heliquinomycin mainly targets actions of the replicative DNA helicases. Gel-retardation experiment indicates that heliquinomycin binds to single-stranded DNA. The single-stranded DNA-binding ability of MCM4/6/7 was affected in the presence of heliquinomycin. The data suggest that heliquinomycin inhibits the DNA helicase activity of MCM4/6/7 complex by stabilizing its interaction with single-stranded DNA.     

宫颈癌组织中MCM5和P16^INK4A mRNA的表达及其临床意义

目的：检测宫颈癌组织中微小染色体维持蛋白-5（minichromosome maintenance protein 5,MCM5）与P16^INK4AmRNA的表达,并探讨其在宫颈癌中的临床意义。方法：采用实时荧光定量PCR（real-time PCR）检测40例宫颈鳞状细胞癌、15例低度宫颈上皮内瘤变（CINⅠ）、20例高度宫颈上皮内瘤变（CINⅡ-Ⅲ）中MCM5和P16^INK4AmRNA的相对表达量,并以20例正常宫颈组织作为对照,分析其与宫颈癌临床病理特征的关系。结果：（1）随着宫颈病变程度的加重,MCM5和P16^INK4AmRNA的表达量逐渐增高。宫颈癌组织中MCM5和P16^INK4AmRNA的表达量分别是正常宫颈组织的（3.026±1.210）倍和（2.540±0.718）倍,差异具有统计学意义（P〈0.05）。宫颈癌组织中MCM5 mRNA的表达量明显高于CINⅠ、CINⅡ-Ⅲ（P〈0.05）,CINⅠ、CINⅡ-Ⅲ中MCM5 mRNA的表达量均显著高于正常宫颈组织,差异具有统计学意义（P〈0.05）,而CINⅠ与CINⅡ-Ⅲ比较差异无统计学意义（P〉0.05）;宫颈癌组织中P16^INK4AmRNA的表达量为正常宫颈组织的（2.54±0.86）倍,差异有统计学意义（P〈0.05）,亦显著高于CINⅠ,差异具有统计学意义（P〈0.05）,但与CINⅡ-Ⅲ比较差异无统计学意义（P〉0.05）。（2）在宫颈癌组织中,MCM5 mRNA的表达量与肿瘤的临床期别、分化程度显著相关（P〈0.01）,但与患者的年龄无关（P〉0.05）;P16^INK4AmRNA的表达量与肿瘤的临床期别、年龄均无关（P〉0.05）,但与肿瘤的分化程度相关（P〈0.01）。结论：MCM5、P16^INK4A的高表达可能在宫颈癌的发展中起重要作用。MCM5基因检测有助于区分癌前病变和宫颈癌,有望成为宫颈癌肿瘤增生的新标志物。P16^INK4A的检测在宫颈病变筛查中具有重要意义,有助于CIN的分级并预测转归,从而提高宫颈癌筛查率。     

Incremental Genetic Perturbations to MCM2-7 Expression and Subcellular Distribution Reveal Exquisite Sensitivity of Mice to DNA Replication Stress

Mutations causing replication stress can lead to genomic instability (GIN). In vitro studies have shown that drastic depletion of the MCM2-7 DNA replication licensing factors, which form the replicative helicase, can cause GIN and cell proliferation defects that are exacerbated under conditions of replication stress. To explore the effects of incrementally attenuated replication licensing in whole animals, we generated and analyzed the phenotypes of mice that were hemizygous for Mcm2, 3, 4, 6, and 7 null alleles, combinations thereof, and also in conjunction with the hypomorphic Mcm4^Chaos3 cancer susceptibility allele. Mcm4^{Chaos3/Chaos3} embryonic fibroblasts have ∼40% reduction in all MCM proteins, coincident with reduced Mcm2-7 mRNA. Further genetic reductions of Mcm2, 6, or 7 in this background caused various phenotypes including synthetic lethality, growth retardation, decreased cellular proliferation, GIN, and early onset cancer. Remarkably, heterozygosity for Mcm3 rescued many of these defects. Consistent with a role in MCM nuclear export possessed by the yeast Mcm3 ortholog, the phenotypic rescues correlated with increased chromatin-bound MCMs, and also higher levels of nuclear MCM2 during S phase. The genetic, molecular and phenotypic data demonstrate that relatively minor quantitative alterations of MCM expression, homeostasis or subcellular distribution can have diverse and serious consequences upon development and confer cancer susceptibility. The results support the notion that the normally high levels of MCMs in cells are needed not only for activating the basal set of replication origins, but also “backup” origins that are recruited in times of replication stress to ensure complete replication of the genome.     

Regulation of the Minichromosome Maintenance Protein 3 (MCM3) Chromatin Binding by the Prolyl Isomerase Pin1

Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs.Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2–7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.     

Interactions of MCP1 with components of the replication machinery in mammalian cells

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.     

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

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

A potential role for mini-chromosome maintenance (MCM) proteins in initiation at the dihydrofolate reductase replication origin.

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.     

Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.     

Protein kinase A is required for chromosomal DNA replication.

Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.     

Identification of mini‐chromosome maintenance 8 as a potential prognostic marker and its effects on proliferation and apoptosis in gastric cancer

Mini‐chromosome maintenance (MCM) proteins play important roles in initiating eukaryotic genome replication. The MCM family of proteins includes several members associated with the development and progression of certain cancers. We performed online data mining to assess the expression of MCMs in gastric cancer (GC) and the correlation between their expression and survival in patients with GC. Notably, MCM8 expression was undoubtedly up‐regulated in GC, and higher expression correlated with shorter overall survival (OS) and progression‐free survival (PFS) in patients with GC. However, the role of MCM8 in GC has not been previously explored. Our in vitro experiments revealed that MCM8 knockdown inhibited cell growth and metastasis. Moreover, MCM8 knockdown induced apoptosis. Mechanistically, the expression levels of Bax and cleaved caspase‐3 were increased, whereas Bcl‐2 expression decreased. Additionally, we demonstrated that MCM8 knockdown suppressed tumorigenesis in vivo. Overall, these results suggest that MCM8 plays a significant role in GC progression.     

Interaction between RAD51 and MCM complex is essential for RAD51 foci forming in colon cancer HCT116 cells

Colon cancer remains one of the most common digestive system malignancies in the World. This study investigated the possible interaction between RAD51 and minichromosome maintenance proteins (MCMs) in HCT116 cells, which can serve as a model system for forming colon cancer foci. The interaction between RAD51 and MCMs was detected by mass spectrometry. Silenced MCM vectors were transfected into HTC116 cells. The expressions of RAD51 and MCMs were detected using Western blotting. Foci forming and chromatin fraction of RAD51 in HCT116 cells were also analyzed. The results showed that RAD51 directly interacted with MCM2, MCM3, MCM5, and MCM6 in colon cancer HTC116 cells. Suppression of MCM2 or MCM6 by shRNA decreased the chromatin localization of RAD51 in HTC116 cells. Moreover, silenced MCM2 or MCM6 decreased the foci forming of RAD51 in HTC116 cells. Our study suggests that the interaction between MCMs and RAD51 is essential for the chromatin localization and foci forming of RAD51 in HCT116 cell DNA damage recovery, and it may be a theoretical basis for analysis of RAD51 in tumor samples of colon cancer patients.     

Ancient diversification of eukaryotic MCM DNA replication proteins

Background  

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

Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G(1) and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G(1). MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G(2), reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G(2), at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G(1) followed by a recruitment to multiple sites in chromatin subsequent to replication.