In silico functional, structural and pathogenicity analysis of missense single nucleotide polymorphisms in human MCM6 gene.

Single nucleotide polymorphisms (SNPs) are one of the most common determinants and potential biomarkers of human disease pathogenesis. SNPs could alter amino acid residues, leading to the loss of structural and functional integrity of the encoded protein. In humans, members of the minichromosome maintenance (MCM) family play a vital role in cell proliferation and have a significant impact on tumorigenesis. Among the MCM members, the molecular mechanism of how missense SNPs of minichromosome maintenance complex component 6 (MCM6) contribute to DNA replication and tumor pathogenesis is underexplored and needs to be elucidated. Hence, a series of sequence and structure-based computational tools were utilized to determine how mutations affect the corresponding MCM6 protein. From the dbSNP database, among 15,009 SNPs in the MCM6 gene, 642 missense SNPs (4.28%), 291 synonymous SNPs (1.94%), and 12,500 intron SNPs (83.28%) were observed. Out of the 642 missense SNPs, 33 were found to be deleterious during the SIFT analysis. Among these, 11 missense SNPs (I123S, R207C, R222C, L449F, V456M, D463G, H556Y, R602H, R633W, R658C, and P815T) were found as deleterious, probably damaging, affective and disease-associated. Then, I123S, R207C, R222C, V456M, D463G, R602H, R633W, and R658C missense SNPs were found to be highly harmful. Six missense SNPs (I123S, R207C, V456M, D463G, R602H, and R633W) had the potential to destabilize the corresponding protein as predicted by DynaMut2. Interestingly, five high-risk mutations (I123S, V456M, D463G, R602H, and R633W) were distributed in two domains (PF00493 and PF14551). During molecular dynamics simulations analysis, consistent fluctuation in RMSD and RMSF values, high Rg and hydrogen bonds in mutant proteins compared to wild-type revealed that these mutations might alter the protein structure and stability of the corresponding protein. Hence, the results from the analyses guide the exploration of the mechanism by which these missense SNPs of the MCM6 gene alter the structural integrity and functional properties of the protein, which could guide the identification of ways to minimize the harmful effects of these mutations in humans.

High MCM6 expression promotes proliferation and correlates with poor prognosis in triple-negative breast cancer.

OBJECTIVE: Triple-negative breast cancer (TNBC) is an aggressive subtype with a poor prognosis. Minichromosome maintenance genes (MCM2-7) crucial for DNA replication are significant biomarkers for various tumor types; however, their roles in TNBC remain underexplored. MATERIALS AND METHODS: We utilized four TNBC-related GEO databases to examine MCM2-7 gene expression and predict its prognosis in TNBC, performing single-cell analysis and GSEA to discover MCM6's potential function. The Cancer Dependency Map gene effect scores and CCK8 assay were used to assess MCM6's impact on TNBC cell proliferation. The correlations between MCM6 expression, immune infiltrates, and immune cells were also analyzed. WGCNA and LASSO Cox regression built a risk score model predicting TNBC patient survival based on MCM6-related gene expression. RESULTS: MCM2-7 gene expression was higher in TNBC tissues compared to adjacent normal tissues. High MCM6 expression correlated with shorter TNBC patient survival time. GSEA and single-cell analysis revealed a relationship between elevated MCM6 expression and the cell cycle pathway. MCM6 knockdown inhibited TNBC cell proliferation. A risk model featuring MCM6, CDC23, and CCNB1 effectively predicts TNBC patient survival. CONCLUSIONS: MCM6 overexpression in TNBC links to a worse prognosis and reduced cell proliferation upon MCM6 knockdown. We developed a risk score model based on MCM6-related genes predicting TNBC patient prognosis, potentially assisting future treatment strategies.

Genome-wide binding sites of Plasmodium falciparum mini chromosome maintenance protein MCM6 show new insights into parasite DNA replication.

Multiple rounds of DNA replication take place in various stages of the life cycle in the human malaria parasite Plasmodium falciparum. Previous bioinformatics analysis has shown the presence of putative Autonomously Replicating Sequence (ARS) like sequences in the Plasmodium genome. However, the actual sites and frequency of replication origins in the P. falciparum genome based on experimental data still remain elusive. Minichromosome maintenance (MCM) proteins are recruited by the Origin recognition complex (ORC) to the origins of replication in eukaryotes including P. falciparum. We used PfMCM6 for chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the quest for identification of putative replication origins in the parasite. PfMCM6 DNA binding sites annotation revealed high enrichment at exon regions. This is contrary to higher eukaryotes that show an inclination of origin sites towards transcriptional start sites. ChIP-seq results were further validated by ChIP-qPCR results as well as nascent strand abundance assay at the selected PfMCM6 enriched sites that also showed preferential binding of PfORC1 suggesting potential of these sites as origin sites. Further, PfMCM6 ChIP-seq data showed a positive correlation with previously published histone H4K8Ac genome-wide binding sites but not with H3K9Ac sites suggesting epigenetic control of replication initiation sites in the parasites. Overall, our data show the genome-wide distribution of PfMCM6 binding sites with their potential as replication origins in this deadly human pathogen that not only broadens our knowledge of parasite DNA replication and its unique biology, it may help to find new avenues for intervention processes.

De novo MCM6 variants in neurodevelopmental disorders: a recognizable phenotype related to zinc binding residues.

The minichromosome maintenance (MCM) complex acts as a DNA helicase during DNA replication, and thereby regulates cell cycle progression and proliferation. In addition, MCM-complex components localize to centrosomes and play an independent role in ciliogenesis. Pathogenic variants in genes coding for MCM components and other DNA replication factors have been linked to growth and developmental disorders as Meier-Gorlin syndrome and Seckel syndrome. Trio exome/genome sequencing identified the same de novo MCM6 missense variant p.(Cys158Tyr) in two unrelated individuals that presented with overlapping phenotypes consisting of intra-uterine growth retardation, short stature, congenital microcephaly, endocrine features, developmental delay and urogenital anomalies. The identified variant affects a zinc binding cysteine in the MCM6 zinc finger signature. This domain, and specifically cysteine residues, are essential for MCM-complex dimerization and the induction of helicase activity, suggesting a deleterious effect of this variant on DNA replication. Fibroblasts derived from the two affected individuals showed defects both in ciliogenesis and cell proliferation. We additionally traced three unrelated individuals with de novo MCM6 variants in the oligonucleotide binding (OB)-fold domain, presenting with variable (neuro)developmental features including autism spectrum disorder, developmental delay, and epilepsy. Taken together, our findings implicate de novo MCM6 variants in neurodevelopmental disorders. The clinical features and functional defects related to the zinc binding residue resemble those observed in syndromes related to other MCM components and DNA replication factors, while de novo OB-fold domain missense variants may be associated with more variable neurodevelopmental phenotypes. These data encourage consideration of MCM6 variants in the diagnostic arsenal of NDD.

MCM6 promotes intrahepatic cholangiocarcinoma progression by upregulating E2F1 and enhancing epithelial-mesenchymal transition.

Minichromosome maintenance complex component 6 (MCM6), a member of the MCM family, plays a pivotal role in DNA replication initiation and genome duplication of proliferating cells. MCM6 is upregulated in multiple malignancies and is considered a novel diagnostic biomarker. However, the functional contributions and prognostic value of MCM6 in intrahepatic cholangiocarcinoma (ICC) remain unexplored. In this study, we investigated the molecular function of MCM6 in ICC. Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO, GSE107943) indicated an upregulation of MCM6 in tumor tissues. Immunohistochemical analysis performed on 115 cases of ICC samples confirmed the upregulation of MCM6 and further suggested that a high level of MCM6 expression predicted shorter overall and disease-free survival in ICC patients. Functional studies suggested that MCM6 knockdown significantly suppressed cell viability, blocked cell cycle progression and inhibited metastasis, while the enhancement of MCM6 expression promoted the proliferation and migration of ICC cells both in vitro and in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) suggested that the epithelial-mesenchymal transition (EMT) and E2F1-correlated genes were enriched in ICC tissues with high MCM6 expression. Further verification indicated that MCM6 promoted the EMT of ICC cells via upregulating E2F1. In addition, E2F1 knockdown partially blocked the pro-malignant effects of MCM6 overexpression. In summary, MCM6 was found to be a novel prognostic and predictive marker for ICC. MCM6 promoted ICC progression via activation of E2F1-mediated EMT.

MCM6 is a critical transcriptional target of YAP to promote gastric tumorigenesis and serves as a therapeutic target.

Rationale: Hyperactivation of Hippo-Yes-associated protein (YAP) signaling pathway governs tumorigenesis of gastric cancer (GC). Here we reveal that minichromosome maintenance complex component 6 (MCM6) is a critical transcriptional target of YAP in GC. We aim to investigate the function, mechanism of action, and clinical implication of MCM6 in GC. Methods: The downstream targets of YAP were screened by RNA sequencing (RNA-seq) and microarray, and further validated by chromatin immunoprecipitation PCR and luciferase reporter assays. The clinical implication of MCM6 was assessed in multiple GC cohorts. Biological function of MCM6 was evaluated in vitro, in patient-derived organoids, and in vivo. RNA-seq was performed to unravel downstream signaling of MCM6. Potential MCM6 inhibitor was identified and the effect of MCM6 inhibition on GC growth was evaluated. Results: Integrative RNA sequencing and microarray analyses revealed MCM6 as a potential YAP downstream target in GC. The YAP-TEAD complex bound to the promoter of MCM6 to induce its transcription. Increased MCM6 expression was commonly observed in human GC tissues and predicted poor patients survival. MCM6 knockdown suppressed proliferation and migration of GC cells and patient-derived organoids, and attenuated xenograft growth and peritoneal metastasis in mice. Mechanistically, MCM6 activated PI3K/Akt/GSK3ß signaling to support YAP-potentiated gastric tumorigenicity and metastasis. Furthermore, MCM6 deficiency sensitized GC cells to chemo- or radiotherapy by causing DNA breaks and blocking ATR/Chk1-mediated DNA damage response (DDR), leading to exacerbated cell death and tumor regression. As there are no available MCM6 inhibitors, we performed high-throughput virtual screening and identified purpureaside C as a novel MCM6 inhibitor. Purpureaside C not only suppressed GC growth but also synergized with 5-fluorouracil to induce cell death. Conclusions: Hyperactivated YAP in GC induces MCM6 transcription via binding to its promoter. YAP-MCM6 axis facilitates GC progression by inducing PI3K/Akt signaling. Targeting MCM6 suppresses GC growth and sensitizes GC cells to genotoxic agents by modulating ATR/Chk1-dependent DDR, providing a promising strategy for GC treatment.

MCM6 Promotes Hepatocellular Carcinoma Progression via the Notch Pathway: Clinical, Functional, and Genomic Insights.

Objective: To evaluate the expression profile of MCM6 in HCC and the relationship between MCM6 level and clinicopathological parameters through bioinformatics analysis of several databases. Methods: MCM expression level, clinical parameters, survival data, and gene set enrichment analysis were analyzed by bioinformatics database, including Oncomine™, UALCAN, HCCDB, TCGA, cBioPortal, and LinkedOmics. Real-time PCR, western blotting, and IHC staining were conducted to identify the expression of MCM6 in HCC compared to normal liver tissues. Results: Bioinformatics analysis indicated that the mRNA of MCM6 was obviously increased in multiple cancer types, especially in HCC. MCM6 level was positively associated with multiple clinical parameters (stage 3 and grades 3 and 4) and negatively associated with patient outcomes (overall survival). Moreover, enrichment of functions and signaling pathways analysis of MCM6 suggested that MCM6 might mediate DNA replication and cellular metabolism to promote the development and progression of HCC. Furthermore, IHC staining and western blotting indicated that the MCM6 was enhanced in HCC tissue, and MCM6 could promote HCC proliferation in activating Notch pathway via WB and bioinformatic analysis. Conclusion: This study actually revealed the expression and related functions of MCM6 in HCC. Furthermore, MCM6 is a carcinogenic role in activating Notch pathway to promote HCC cell proliferation, which may be a new prognostic biomarker and therapeutic target for HCC patients.

Bioinformatics Analysis Reveals MCM3 as an Important Prognostic Marker in Cervical Cancer.

The minichromosome maintenance complex 3 (MCM3) is essential for the regulation of DNA replication and cell cycle progression. However, the expression and prognostic values of MCM3 in cervical cancer (CC) have not been well-studied. Herein, we investigated the expression patterns and survival data of MCM3 in cervical cancer patients from the ONCOMINE, GEPIA, Human Protein Atlas, UALCAN, Kaplan-Meier Plotter, and LinkedOmics databases. The expression level of MCM3 is negatively correlated with advanced tumor stage and metastatic status. Specifically, MCM3 is significantly differentially expressed between patients in stage 1 and stage 3 cervical cancer with p value 0.0138. Similarly, the p values between stage 1 and stage 4 cervical cancer, between stage 2 and stage 3, and between stage 2 and stage 4 are 0.00089, 0.0244, and 0.00197, respectively. Not only that, cervical cancer patients with high mRNA expression of MCM3 may indicate longer overall survival but indicate shorter relapse-free survival. PRIM2 and MCM6 are positively correlated genes of MCM3. Bioinformatics analysis revealed that MCM3 might be considered a biological indicator for prognostic evaluation of cervical cancer. However, it is currently limited to bioinformatics analysis, and more clinical tissue specimens and cell experiments are needed to further explore the role of MCM3 in the occurrence and progression of cervical cancer.

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii.

DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions ('multiple fission'). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom.

Methylation-dependent MCM6 repression induced by LINC00472 inhibits triple-negative breast cancer metastasis by disturbing the MEK/ERK signaling pathway.

Long noncoding RNAs (lncRNAs) have been identified to be dysregulated in multiple cancer types, which are speculated to be of vital significance in regulating several hallmarks of cancer biology. Triple-negative breast cancer (TNBC) is acknowledged as an aggressive subtype of breast cancer. In this study, we found the lncRNA LINC00472 was poorly expressed in TNBC tissues and cells. Overexpression of LINC00472 could inhibit the proliferation, invasion and migration of MDA-MB-231 cells. On the contrary, minichromosome maintenance complex component 6 (MCM6) was highly expressed in TNBC tissues and MDA-MB-231 cells due to suppressed methylation. LINC00472 induced site-specific DNA methylation and reduced the MCM6 expression by recruiting DNA methyltransferases into the MCM6 promoter. Since the restoration of MCM6 weakened the tumor-suppressive effect of LINC00472 on MDA-MB-231 cells, LINC00472 potentially acted as a tumor suppressor by inhibiting MCM6. In addition, in vivo experiments further substantiated that overexpression of LINC00472 inhibited tumor growth and metastasis to lungs by decreasing the expression of MCM6. Overall, the present study demonstrated that LINC00472-mediated epigenetic silencing of MCM6 contributes to the prevention of tumorigenesis and metastasis in TNBC, providing an exquisite therapeutic target for TNBC.

Role of Amygdalin in Blocking DNA Replication in Breast Cancer In Vitro.

BACKGROUND: Amygdalin has anticancer benefits because of its active component, hydrocyanic acid. However, the underlying molecular mechanism is unclear. OBJECTIVE: This study aimed to investigate the molecular mechanism by which amygdalin exerts antiproliferative effects in the human Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line. METHODS: MCF-7 cells were exposed to amygdalin at a particular IC50 value for 24 and 48 hours and compared to non-treated cells. An Affymetrix whole-transcript expression array was used to analyze the expression of 32 genes related to DNA replication. RESULTS: Among the 32 genes, amygdalin downregulated the expression of 16 genes and 19 genes by >1.5-fold at 24 and 48 hours, respectively. At 24 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, PRIM1, and PRIM2; DNA polymerase δ complex: POLD3; DNA polymerase ε complex: POLE4, Minichromosome Maintenance protein (MCM) complex (helicase): MCM2, MCM3, MCM4, MCM6, and MCM7; clamp and clamp loader: PCNA; nuclease: FEN1; and DNA ligase: LIG1. At 48 hours, the downregulated genes from the DNA polymerase α-primase complex were POLA1, POLA2, and PRIM1; DNA polymerase δ complex: POLD3; DNA polymerase ε complex: POLE and POLE2; MCM complex (helicase): MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7; clamp and clamp loader: PCNA, RFC2, and RFC3; RNase H: RNASEH2A; nucleases: DNA2 and FEN1; and DNA ligase: LIG1. CONCLUSION: Amygdalin treatment caused downregulation of several genes that play critical roles in DNA replication in the MCF-7 cell line. Thus, it might be useful as an anticancer agent.

The DNA replication regulator MCM6: An emerging cancer biomarker and target.

MCM6 is a significant DNA replication regulator that plays a crucial role in sustaining the cell cycle. In many cancer cells, MCM6 expression is enhanced. For example, persistently increased expression of MCM6 promotes the formation, development and progression of hepatocellular carcinoma (HCC). Up- and down-regulation studies have indicated that MCM6 regulates cell cycle, proliferation, metastasis, immune response and the maintenance of the DNA replication system. MCM6 can also regulate downstream signaling such as MEK/ERK thus promoting carcinogenesis. Accordingly, MCM6 may represent a sensitive and specific biomarker to predict adverse progression and poor outcome. Furthermore, inhibition of MCM6 may be an effective cancer treatment. The present review summarizes the latest results on the inactivating and activating functions of MCM6, underlining its function in carcinogenesis. Further studies of the carcinogenic functions of MCM6 may provide novel insight into cancer biology and shed light on new approaches for cancer diagnosis and treatment.

Minichromosome maintenance 6 complex component identified by bioinformatics analysis and experimental validation in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC), the main subtype of esophageal cancer (EC), is a common lethal type of cancer with a high mortality rate. The aim of the present study was to select key relevant genes and identify potential mechanisms involved in the development of ESCC based on bioinformatics analysis. Minichromosome maintenance 6 complex component (MCM6) has been identified to be upregulated in multiple malignancies; however, its contributions to ESCC remain unclear. For the purposes of the present study, four datasets were downloaded from the Gene Expression Omnibus (GSE63941, GSE26886, GSE17351 and GSE77861), and the intersection of the differentially expressed genes was obtained using a Venn diagram. The protein­protein interaction was then constructed, and the modules were verified by Cytoscape, in which the key genes have a high connectivity degree with other genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway were subsequently filtered out to analyze the development of ESCC. MCM6, an upregulated gene, was selected and connected with most of the other genes, for further research validation. The expression levels of MCM6 were then assessed using the Oncomine, GEPIA and UALCAN databases and validated in both ESCC tissues samples and cell lines by immunohistochemistry and RT­qPCR. Cell counting kit­8 (CCK­8), flow cytometry, wound healing and Transwell assays were used to determine the proliferation, apoptosis, cell cycle, migration and invasion of ESCC cells. A total of 24 genes were identified by a series of bioinformatics analyses and the results revealed that the genes were associated with DNA replication and cell cycle. Experimental validation revealed that MCM6 expression was significantly elevated in both ESCC tissues and cell lines. The results were consistent with those of bioinformatics analysis. Furthermore, the knockdown of MCM6 inhibited cell proliferation, migration and invasion and promoted cell apoptosis, and made cells arrested in S stage. In summary, the findings of bioinformatics analysis provided a novel hypothesis for ESCC progression. In particular, the aberrantly elevated expression of MCM6 is a potential biomarker for ESCC diagnosis and treatment.

The impact of MCM6 on hepatocellular carcinoma in a Southern Chinese Zhuang population.

Minichromosome maintenance complex component 6 (MCM6) is involved in tumorigenesis of hepatocellular carcinoma (HCC). Because its effect on different populations remains unclear, this study investigated the impact of MCM6 on HCC in Southern Chinese Zhuang population. In addition to assessing the global mRNA levels of MCM6 based on The Cancer Genome Atlas database (TCGA) and The Gene Expression Omnibus database (GEO), associations between MCM6 mRNA levels and clinicopathological features were analyzed. High MCM6 levels were associated with high alpha-fetoprotein (AFP) (>20 ng/mL in serum) (P < 0.0001) and advanced clinical stage (III + IV) (P < 0.001). Higher MCM6 was associated with poorer outcomes (P < 0.01) in these databases. Furthermore, the mRNA and protein expression of MCM6 in the Guangxi Zhuang population was detected by quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry (IHC). The results showed that MCM6 levels were up-regulated in the Zhuang population with HCC. Higher MCM6 protein levels were correlated with larger tumor size (>5 cm) (P = 0.038) and advanced clinical stage (III + IV) (p = 0.023). Bioinformatic enrichment analysis of MCM6 and its interacting proteins (CDT1,WEE1,TRIM28 and MKI67) suggested that in addition to being involved in the cell cycle process, these complexes could also be involved in protein binding, pre-replication complex assemble, and nucleus metabolism. Based on the protein-protein interaction (PPI) network with module screen, the interactions between MCM6 and its potential interacting proteins were further studied through protein docking with hot spot analysis. Additionally, the results of the algorithms combining the ROC of MCM6 and its interacting proteins showed that combination biomarker analysis has better HCC diagnosis ability than the single MCM6 test. The combination of MCM6 and TRIM28 was more suitable for the Guangxi Zhuang population. Overall, our study suggests that MCM6 plays an important role in the growth of HCC. MCM6 could be an optimal biomarker for diagnosing HCC and a potential molecular target for HCC therapy in the Zhuang population.

A novel transcriptional cascade is involved in Fzr-mediated endoreplication.

Endoreplication, known as endocycle, is a variant of the cell cycle that differs from mitosis and occurs in specific tissues of different organisms. Endoreplicating cells generally undergo multiple rounds of genome replication without chromosome segregation. Previous studies demonstrated that Drosophila fizzy-related protein (Fzr) and its mammalian homolog Cdh1 function as key regulators of endoreplication entrance by activating the anaphase-promoting complex/cyclosome to initiate the ubiquitination and subsequent degradation of cell cycle factors such as Cyclin B (CycB). However, the molecular mechanism underlying Fzr-mediated endoreplication is not completely understood. In this study, we demonstrated that the transcription factor Myc acts downstream of Fzr during endoreplication in Drosophila salivary gland. Mechanistically, Fzr interacts with chromatin-associated histone H2B to enhance H2B ubiquitination in the Myc promoter and promotes Myc transcription. In addition to negatively regulating CycB transcription, the Fzr-ubiquitinated H2B (H2Bub)-Myc signaling cascade also positively regulates the transcription of the MCM6 gene that is involved in DNA replication by directly binding to specific motifs within their promoters. We further found that the Fzr-H2Bub-Myc signaling cascade regulating endoreplication progression is conserved between insects and mammalian cells. Altogether, our work uncovers a novel transcriptional cascade that is involved in Fzr-mediated endoreplication.

DNA unwinding mechanism of a eukaryotic replicative CMG helicase.

High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.

An approximate full-likelihood method for inferring selection and allele frequency trajectories from DNA sequence data.

Most current methods for detecting natural selection from DNA sequence data are limited in that they are either based on summary statistics or a composite likelihood, and as a consequence, do not make full use of the information available in DNA sequence data. We here present a new importance sampling approach for approximating the full likelihood function for the selection coefficient. Our method CLUES treats the ancestral recombination graph (ARG) as a latent variable that is integrated out using previously published Markov Chain Monte Carlo (MCMC) methods. The method can be used for detecting selection, estimating selection coefficients, testing models of changes in the strength of selection, estimating the time of the start of a selective sweep, and for inferring the allele frequency trajectory of a selected or neutral allele. We perform extensive simulations to evaluate the method and show that it uniformly improves power to detect selection compared to current popular methods such as nSL and SDS, and can provide reliable inferences of allele frequency trajectories under many conditions. We also explore the potential of our method to detect extremely recent changes in the strength of selection. We use the method to infer the past allele frequency trajectory for a lactase persistence SNP (MCM6) in Europeans. We also infer the trajectory of a SNP (EDAR) in Han Chinese, finding evidence that this allele's age is much older than previously claimed. We also study a set of 11 pigmentation-associated variants. Several genes show evidence of strong selection particularly within the last 5,000 years, including ASIP, KITLG, and TYR. However, selection on OCA2/HERC2 seems to be much older and, in contrast to previous claims, we find no evidence of selection on TYRP1.

Changes in MCM2-7 proteins at senescence.

Cellular aging is characterized by the loss of DNA replication capability and is mainly brought about by various changes in chromatin structure. Here, we examined changes in MCM2-7 proteins, which act as a replicative DNA helicase, during aging of human WI38 fibroblasts at the single-cell level. We used nuclear accumulation of p21 as a marker of senescent cells, and examined changes in MCM2-7 by western blot analysis. First, we found that senescent cells are enriched for cells with a DNA content higher than 4N. Second, the levels of MCM2, MCM3, MCM4 and MCM6 proteins decreased in senescent cells. Third, cytoplasmic localization of MCM2 and MCM7 was observed in senescent cells, from an analysis of MCM2-7 except for MCM5. Consistent with this finding, fragmented MCM2 was predominant in these cells. These age-dependent changes in MCM2-7, a protein complex that directly affects cellular DNA replication, may play a critical role in cellular senescence.

Comparison of Lactase Variant MCM6 -13910 C>T Testing and Self-report of Dairy Sensitivity in Patients With Irritable Bowel Syndrome.

GOALS: To evaluate agreement of MCM6-13910 with self-report of dairy sensitivity (DS) and lactose hydrogen methane breath test (LHMBT) results in subjects with irritable bowel syndrome (IBS). BACKGROUND: IBS is a functional gastrointestinal disorder with symptoms including abdominal pain, variable bowel habits, and bloating. Adult patients with lactose malabsorption may present with similar symptoms. Patients with lactose malabsorption have a lactase nonpersistent (LNP) phenotype. Recent studies found 2 single nucleotide polymorphisms associated with LNP: G/A-22018 and C/T-13910. STUDY: Genotyping the MCM6-13910 variant of LNP in 538 IBS patients and 317 controls (without IBS). Subjects completed questionnaires pertaining to gastrointestinal problems and dietary consumption, with charts abstracted. RESULTS: Self-reported DS was higher in IBS (45%) than controls (9.8%, odds ratio=6.46, P<0.001). The C/C-13910 genotype was similar in IBS cases and controls, 81 (15.1%) and 47 (14.8%). Among subjects reporting DS, 49 (18.0%) had the C/C genotype. Overall agreement between genotype and self-reported DS was 0.06 in IBS and 0.07 in controls. There were 20 subjects with LHMBT results; 3 had positive results, 17 were negative. LNP genotypes were found in all 3 of positive LHMBT results; 16 had negative LHMBT among the 17 who were lactase persistent. Agreement between C/C-13910 genotype and LHMBT was excellent with κ-statistic of 0.83 (0.50-1.00). CONCLUSIONS: In IBS patients, self-report of lactose intolerance are highly prevalent but are a poor indicator of underlying C/C-13910 genotype. LHMBT had excellent agreement with C/C-13910 genotype.

GTSE1, CDC20, PCNA, and MCM6 Synergistically Affect Regulations in Cell Cycle and Indicate Poor Prognosis in Liver Cancer.

GTSE1 is well correlated with tumor progression; however, little is known regarding its role in liver cancer prognosis. By analyzing the hepatocellular carcinoma (HCC) datasets in GEO and TCGA databases, we showed that high expression of GTSE1 was correlated with advanced pathologic stage and poor prognosis of HCC patients. To investigate underlying molecular mechanism, we generated GTSE1 knockdown HCC cell line and explored the effects of GTSE1 deficiency in cell growth. Between GTSE1 knockdown and wild-type HCC cells, we identified 979 differentially expressed genes (520 downregulated and 459 upregulated genes) in the analysis of microarray-based gene expression profiling. Functional enrichment analysis of DEGs suggested that S phase was dysregulated without GTSE1 expression, which was further verified from flow cytometry analysis. Moreover, three other DEGs: CDC20, PCNA, and MCM6, were also found contributing to GTSE1-related cell cycle arrest and to be associated with poor overall survival of HCC patients. In conclusion, GTSE1, together with CDC20, PCNA, and MCM6, may synergistically promote adverse prognosis in HCC by activating cell cycle. Genes like GTSE1, CDC20, PCNA, and MCM6 may be promising prognostic molecular biomarkers in liver cancer.