Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase.

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate-distinct from that between Mcm2/5 used for origin loading-is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.

Mechanism of eukaryotic origin unwinding is a dual helicase DNA shearing process.

DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA polymerases. In bacteria, origin DNA is presumed to be melted by accessory proteins that allow loading of two ring-shaped replicative helicases around single-strand DNA (ssDNA) for bidirectional unwinding and DNA replication. In eukaryotes, by contrast, two replicative CMG (Cdc45-Mcm2-7-GINS) helicases are initially loaded head to head around origin double-strand DNA (dsDNA), and there does not appear to be a separate origin unwinding factor. This led us to investigate whether head-to-head CMGs use their adenosine triphosphate (ATP)-driven motors to initiate duplex DNA unwinding at the origin. Here, we show that CMG tracks on one strand of the duplex while surrounding it, and this feature allows two head-to-head CMGs to unwind dsDNA by using their respective motors to pull on opposite strands of the duplex. We further show that while CMG is capable of limited duplex unwinding on its own, the extent of unwinding is greatly and rapidly stimulated by addition of the multifunctional CMG-binding protein Mcm10 that is critical for productive initiation of DNA replication in vivo. On the basis of these findings, we propose that Mcm10 is a processivity or positioning factor that helps translate the work performed by the dual CMG motors at the origin into productive unwinding that facilitates bidirectional DNA replication.

Multifaceted role of the DNA replication protein MCM10 in maintaining genome stability and its implication in human diseases.

MCM10 plays a vital role in genome duplication and is crucial for DNA replication initiation, elongation, and termination. It coordinates several proteins to assemble at the fork, form a functional replisome, trigger origin unwinding, and stabilize the replication bubble. MCM10 overexpression is associated with increased aggressiveness in breast, cervical, and several other cancers. Disruption of MCM10 leads to altered replication timing associated with initiation site gains and losses accompanied by genome instability. Knockdown of MCM10 affects the proliferation and migration of cancer cells, manifested by DNA damage and replication fork arrest, and has recently been shown to be associated with clinical conditions like CNKD and RCM. Loss of MCM10 function is associated with impaired telomerase activity, leading to the accumulation of abnormal replication forks and compromised telomere length. MCM10 interacts with histones, aids in nucleosome assembly, binds BRCA2 to maintain genome integrity during DNA damage, prevents lesion skipping, and inhibits PRIMPOL-mediated repriming. It also interacts with the fork reversal enzyme SMARCAL1 and inhibits fork regression. Additionally, MCM10 undergoes several post-translational modifications and contributes to transcriptional silencing by interacting with the SIR proteins. This review explores the mechanism associated with MCM10's multifaceted role in DNA replication initiation, chromatin organization, transcriptional silencing, replication stress, fork stability, telomere length maintenance, and DNA damage response. Finally, we discuss the role of MCM10 in the early detection of cancer, its prognostic significance, and its potential use in therapeutics for cancer treatment.

Investigation into in silico and in vitro approaches for inhibitors targeting MCM10 in Leishmania donovani: a comprehensive study.

Visceral Leishmaniasis (VL), the second neglected tropical disease caused by various Leishmania species, presents a significant public health challenge due to limited treatment options and the absence of vaccines. The agent responsible for visceral leishmaniasis, also referred to as "black fever" in India, is Leishmania donovani. This study focuses on L. donovani Minichromosome maintenance 10 (LdMcm10), a crucial protein in the DNA replication machinery, as a potential therapeutic target in Leishmania therapy using in silico and in vitro approaches. We employed bioinformatics tools, molecular docking, and molecular dynamics simulations to predict potential inhibitors against the target protein. The research revealed that the target protein lacks homologues in the host, emphasizing its potential as a drug target. Ligands from the DrugBank database were screened against LdMcm10 using PyRx software. The top three compounds, namely suramin, vapreotide, and pasireotide, exhibiting the best docking scores, underwent further investigation through molecular dynamic simulation and in vitro analysis. The observed structural dynamics suggested that LdMcm10-ligand complexes maintained consistent binding throughout the 300 ns simulation period, with minimal variations in their backbone. These findings suggest that these three compounds hold promise as potential lead compounds for developing new drugs against leishmaniasis. In vitro experiments also demonstrated a dose-dependent reduction in L. donovani viability for suramin, vapreotide, and pasireotide, with computed IC50 values providing quantitative metrics of their anti-leishmanial efficacy. The research offers a comprehensive understanding of LdMcm10 as a drug target and provides a foundation for further investigations and clinical exploration, ultimately advancing drug discovery strategies for leishmaniasis treatment.

MCM10: An effective treatment target and a prognostic biomarker in patients with uterine corpus endometrial carcinoma.

Molecular profiling has been applied for uterine corpus endometrial carcinoma (UCEC) management for many years. The aim of this study was to explore the role of MCM10 in UCEC and construct its overall survival (OS) prediction models. Data from TCGA, GEO, cbioPotal and COSMIC databases and the methods, such as GO, KEGG, GSEA, ssGSEA and PPI, were employed to bioinformatically detect the effects of MCM10 on UCEC. RT-PCR, Western blot and immunohistochemistry were used to validate the effects of MCM10 on UCEC. Based on Cox regression analysis using the data from TCGA and our clinical data, two OS prediction models for UCEC were established. Finally, the effects of MCM10 on UCEC were detected in vitro. Our study revealed that MCM10 was variated and overexpressed in UCEC tissue and involved in DNA replication, cell cycle, DNA repair and immune microenvironment in UCEC. Moreover, silencing MCM10 significantly inhibited the proliferation of UCEC cells in vitro. Importantly, based on MCM10 expression and clinical features, the OS prediction models were constructed with good accuracy. MCM10 could be an effective treatment target and a prognostic biomarker for UCEC patients. The OS prediction models might help establish the strategies of follow-up and treatment for UCEC patients.

Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression.

The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, GINS) tightly binds Mcm10, an essential replication protein in all eukaryotes. Here we show that Mcm10 has a potent strand-annealing activity both alone and in complex with CMG. CMG-Mcm10 unwinds and then reanneals single strands soon after they have been unwound in vitro. Given the DNA damage and replisome instability associated with loss of Mcm10 function, we examined the effect of Mcm10 on fork regression. Fork regression requires the unwinding and pairing of newly synthesized strands, performed by a specialized class of ATP-dependent DNA translocases. We show here that Mcm10 inhibits fork regression by the well-known fork reversal enzyme SMARCAL1. We propose that Mcm10 inhibits the unwinding of nascent strands to prevent fork regression at normal unperturbed replication forks, either by binding the fork junction to form a block to SMARCAL1 or by reannealing unwound nascent strands to their parental template. Analysis of the CMG-Mcm10 complex by cross-linking mass spectrometry reveals Mcm10 interacts with six CMG subunits, with the DNA-binding region of Mcm10 on the N-face of CMG. This position on CMG places Mcm10 at the fork junction, consistent with a role in regulating fork regression.

Prognostic significance and function of MCM10 in human hepatocellular carcinoma.

Aim: To investigate the role of MCM10, a conserved replication factor, in hepatocellular carcinoma (HCC). Methods: We used data from 364 HCC patients in the Cancer Genome Atlas database and conducted in vitro experiments to confirm the role of MCM10. Results: High MCM10 expression correlated with poor HCC patient outcome and was an independent prognosticator for HCC. Time-dependent receiver operating characteristic curve analysis found that the sequential trend of MCM10 for survival was not inferior to that of the tumor node metastasis stage. The MCM10 model had a higher C-index than the non-MCM10 model, indicating that incorporating MCM10 into a multivariate model improves the model's prognostic accuracy for HCC. Genetic alterations of MCM10 prominently correlated with an unfavorable HCC outcome. Conclusion: Our findings strongly suggest using the MCM10 gene as a prognostic indicator in HCC.

Lay abstract Hepatocellular carcinoma is one of the most aggressive malignant cancers globally. Our study investigated the role of a conserved replication factor, MCM10, in hepatocellular carcinoma. We performed some bioinformatics analysis and in vitro experiments, and successfully found that MCM10 has a good predictive value for survival in patients with hepatocellular carcinoma.

The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing.

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.

Overexpression of MCM10 promotes cell proliferation and predicts poor prognosis in prostate cancer.

BACKGROUND: Prostate cancer (PCa) is one of the most malignant tumors of the male urogenital system. There is an urgent need to identify novel biomarkers for PCa. METHODS: In this study, we evaluated the expression levels of MCM10 in prostate cancer by analyzing public datasets (including The Cancer Genome Atlas and GSE21032). Furthermore, loss of function assays was performed to evaluate the effects of MCM10 on cell proliferation, apoptosis, and colony formation. Furthermore, we performed microarray and bioinformatics analyses to explore the potential mechanisms of MCM10. RESULTS: In the present study, we for the first time revealed MCM10 was significantly upregulated in PCa. Moreover, we found increased MCM10 expression was significantly associated with advanced clinical stage and high Gleason score PCa. Kaplan-Meier analysis demonstrated higher MCM10 expression was associated with a poorer patient prognosis in PCa. Furthermore, loss of function assays showed that MCM10 knockdown inhibited cell proliferation and colony formation, but promoted cell apoptosis. Additionally, we performed microarray and bioinformatics analysis and found MCM10 regulated PCa progression by regulating a series of biological processes including cancer, cell death, and apoptosis. CONCLUSIONS: These results suggest that MCM10 may be a potential diagnostic and therapeutic target for PCa.

Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex.

Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly ("G1-like") and high affinity recruitment when CMG assembly takes place ("S-phase-like"). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus.

Overexpression of minichromosome maintenance protein 10 in medulloblastoma and its clinical implications.

BACKGROUND: Overexpression of minichromosome maintenance (MCM) proteins 2, 3, and 7 is associated with migration and invasion in medulloblastoma (MB). However, expression profiling of all prereplication complex (pre-RC) has not been addressed in MBs. PROCEDURE: We performed mRNA expression profiling of a large set of pre-RC elements in cell lines and tumor tissues of MB. RNAi technology was employed for functional studies in MB cell lines. RESULTS: Our data showed that most of the pre-RC components are significantly overexpressed in MB. Among all pre-RC mRNAs, MCM10 showed the highest level of expression (∼500- to 1,000-fold) in MB cell lines and tissues compared to the levels detected in cerebellum. In addition, RNAi silencing of MCM10 caused reduced cell proliferation and cell viability in MB cells. CONCLUSIONS: Taken together, our study reveals that the pre-RC is dysregulated in MB. In addition, MCM10, a member of this complex, is significantly overexpressed in MB and is required for tumor cell proliferation.

MCM10: one tool for all-Integrity, maintenance and damage control.

Minichromsome maintenance protein 10 (Mcm10) is an essential replication factor that is required for the activation of the Cdc45:Mcm2-7:GINS helicase. Mcm10's ability to bind both ds and ssDNA appears vital for this function. In addition, Mcm10 interacts with multiple players at the replication fork, including DNA polymerase-α and proliferating cell nuclear antigen with which it cooperates during DNA elongation. Mcm10 lacks enzymatic function, but instead provides the replication apparatus with an oligomeric scaffold that likely acts in the coordination of DNA unwinding and DNA synthesis. Not surprisingly, loss of Mcm10 engages checkpoint, DNA repair and SUMO-dependent rescue pathways that collectively counteract replication stress and chromosome breakage. Here, we review Mcm10's structure and function and explain how it contributes to the maintenance of genome integrity.

HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest.

Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G2/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G2/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G2/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G2/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP.

iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.

RNF4 prevents genomic instability caused by chronic DNA under-replication.

Eukaryotic genome stability is maintained by a complex and diverse set of molecular processes. One class of enzymes that promotes proper DNA repair, replication and cell cycle progression comprises small ubiquitin-like modifier (SUMO)-targeted E3 ligases, or STUbLs. Previously, we reported a role for the budding yeast STUbL synthetically lethal with sgs1 (Slx) 5/8 in preventing G2/M-phase arrest in a minichromosome maintenance protein 10 (Mcm10)-deficient model of replication stress. Here, we extend these studies to human cells, examining the requirement for the human STUbL RING finger protein 4 (RNF4) in MCM10 mutant cancer cells. We find that MCM10 and RNF4 independently promote origin firing but regulate DNA synthesis epistatically and, unlike in yeast, the negative genetic interaction between RNF4 and MCM10 causes cells to accumulate in G1-phase. When MCM10 is deficient, RNF4 prevents excessive DNA under-replication at hard-to-replicate regions that results in large DNA copy number alterations and severely reduced viability. Overall, our findings highlight that STUbLs participate in species-specific mechanisms to maintain genome stability, and that human RNF4 is required for origin activation in the presence of chronic replication stress.

S-CDK-regulated bipartite interaction of Mcm10 with MCM is essential for DNA replication.

Mcm10 plays an essential role in the activation of replicative helicase CMG through the cell cycle-regulated interaction with the prototype MCM double hexamer in Saccharomyces cerevisiae. In this study, we reported that Mcm10 is phosphorylated by S-phase cyclin-dependent kinases (S-CDKs) at S66, which enhances Mcm10--MCM association during the S phase. S66A single mutation or even deletion of whole N-terminus (a.a. 1-128) only causes mild growth defects. Nevertheless, S66 becomes indispensable in the absence of the Mcm10 C-terminus ((a.a. 463-571), the major MCM-binding domain. Using a two-degron strategy to efficiently deplete Mcm10, we show that mcm10-S66AΔC has a severe defect in proceeding into the S phase. Notably, both lethality and S-phase deficiency can be rescued by artificially tethering mcm10-S66AΔC to MCM. These findings illustrate how the Mcm10-MCM association is regulated as a crucial event in DNA replication initiation.

A critical threshold of MCM10 is required to maintain genome stability during differentiation of induced pluripotent stem cells into natural killer cells.

Natural killer (NK) cell deficiency (NKD) is a rare disease in which NK cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Recently, a patient with NKD was identified carrying compound heterozygous variants of MCM10 (minichromosome maintenance protein 10), an essential gene required for DNA replication, that caused a significant decrease in the amount of functional MCM10. NKD in this patient presented as loss of functionally mature late-stage NK cells. To understand how MCM10 deficiency affects NK cell development, we generated MCM10 heterozygous (MCM10+/-) induced pluripotent stem cell (iPSC) lines. Analyses of these cell lines demonstrated that MCM10 was haploinsufficient, similar to results in other human cell lines. Reduced levels of MCM10 in mutant iPSCs was associated with impaired clonogenic survival and increased genomic instability, including micronuclei formation and telomere erosion. The severity of these phenotypes correlated with the extent of MCM10 depletion. Significantly, MCM10+/- iPSCs displayed defects in NK cell differentiation, exhibiting reduced yields of hematopoietic stem cells (HSCs). Although MCM10+/- HSCs were able to give rise to lymphoid progenitors, these did not generate mature NK cells. The lack of mature NK cells coincided with telomere erosion, suggesting that NKD caused by these MCM10 variants arose from the accumulation of genomic instability including degradation of chromosome ends.

Immune modulation and prognostic significance of MCM10 in pan-cancer: a comprehensive analysis.

BACKGROUND: Oncogenic processes in cancer are often characterized by dysregulation of critical genes. Our study focused on the minichromosome maintenance 10 replication initiation factor (MCM10) gene's expression and its potential diagnostic and prognostic implications in pan-cancer. METHOD: Leveraging large-scale genomic datasets, and experimental validation we embarked on a comprehensive analysis to shed light on the diagnostic and prognostic role of MCM10. RESULTS: Our findings underscore the wide-ranging up-regulation of MCM10 across 24 major cancer types, positioning it as a ubiquitous player in tumorigenesis. Significantly, MCM10 up-regulation was strongly associated with poorer overall survival in Kidney Renal Papillary Cell Carcinoma (KIRP), Liver Hepatocellular Carcinoma (LIHC), and Lung Adenocarcinoma (LUAD), emphasizing its potential as a valuable prognostic marker in these cancers. While genetic mutations often drive oncogenic processes, our mutational analysis revealed the relative stability of MCM10 in KIRP, LIHC, and LUAD. This suggests that epigenetic (hypomethylation) and non-mutational regulatory mechanisms predominantly govern MCM10 expression in these cancer types. Further analyses demonstrated positive correlations between MCM10 expression and immune cell infiltration, particularly CD8+ T cells and CD4+ T cells, offering insights into the gene's influence on the tumor immune microenvironment. Additionally, pathway enrichment analysis highlighted MCM10-associated genes' involvement in crucial signaling pathways, such as the cell cycle, DNA replication, and repair. Exploring the therapeutic potential, we examined important drugs capable of regulating MCM10 expression, opening doors to personalized treatment strategies. CONCLUSION: Our study elucidates the multifaceted roles of MCM10 in KIRP, LIHC, and LUAD. Its pervasive up-regulation, prognostic significance, epigenetic regulation, and influence on the immune microenvironment provide valuable insights into these cancers. This research contributes to the growing body of evidence surrounding MCM10 and invites further investigation, validation, and potential translational efforts to harness its clinical relevance.

Minichromosome maintenance protein 10 (mcm10) regulates hematopoietic stem cell emergence in the zebrafish embryo.

Hematopoietic stem cells (HSCs) guarantee the continuous supply of all blood lineages during life. In response to stress, HSCs are capable of extensive proliferative expansion, whereas in steady state, HSCs largely remain in a quiescent state to prevent their exhaustion. DNA replication is a very complex process, where many factors need to exert their functions in a perfectly concerted manner. Mini-chromosome-maintenance protein 10 (Mcm10) is an important replication factor, required for proper assembly of the eukaryotic replication fork. In this report, we use zebrafish to study the role of mcm10 during embryonic development, and we show that mcm10 specifically regulates HSC emergence from the hemogenic endothelium. We demonstrate that mcm10-deficient embryos present an accumulation of DNA damages in nascent HSCs, inducing their apoptosis. This phenotype can be rescued by knocking down p53. Taken all together, our results show that mcm10 plays an important role in the emergence of definitive hematopoiesis.

RTEL1 and MCM10 overcome topological stress during vertebrate replication termination.

Topological stress can cause converging replication forks to stall during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome fork stalling, suggesting that alternative mechanisms of termination exist. Using proteomics in Xenopus egg extracts, we show that the helicase RTEL1 and the replisome protein MCM10 are highly enriched on chromatin during fork convergence and are crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 cooperate to promote fork convergence and do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 play a general role in promoting progression of stalled forks, including when forks stall during termination. Our data reveal an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.