Search

In the first trimester of pregnancy the human placenta grows rapidly, making it sensitive to changes in the intrauterine environment. To test whether exposure to an environment in utero often associated with obesity modifies placental proteome and function, we performed untargeted proteomics (LC-MS/MS) in placentas from 19 women (gestational age 35-48 days, i.e. 5+0-6+6 weeks). Maternal clinical traits (body mass index, leptin, glucose, C-peptide and insulin sensitivity) and gestational age were recorded. DNA replication and cell cycle pathways were enriched in the proteome of placentas of women with low maternal insulin sensitivity. Driving these pathways were the minichromosome maintenance (MCM) proteins MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7 (MCM-complex). These proteins are part of the pre-replicative complex and participate in DNA damage repair. Indeed, MCM6 and γH2AX (DNA-damage marker) protein levels correlated in first trimester placental tissue (r = 0.514, P<0.01). MCM6 and γH2AX co-localized to nuclei of villous cytotrophoblast cells, the proliferative cell type of the placenta, suggesting increased DNA damage in this cell type. To mimic key features of the intrauterine obesogenic environment, a first trimester trophoblast cell line, i.e., ACH-3P, was exposed to high insulin (10 nM) or low oxygen tension (2.5% O2). There was a significant correlation between MCM6 and γH2AX protein levels, but these were independent of insulin or oxygen exposure. These findings show that chronic exposure in utero to reduced maternal insulin sensitivity during early pregnancy induces changes in the early first trimester placental proteome. Pathways related to DNA replication, cell cycle and DNA damage repair appear especially sensitive to such an in utero environment., (© 2024 The Author(s).)

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses., (© 2024. The Author(s).)

The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCF Dia2 . mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes., (© 2024. The Author(s).)

Eukaryotic DNA replication is a tightly controlled process that occurs in two main steps, i.e., licensing and firing, which take place in the G1 and S phases of the cell cycle, respectively. In Saccharomyces cerevisiae, the budding yeast, replication origins contain consensus sequences that are recognized and bound by the licensing factor Orc1-6, which then recruits the replicative Mcm2-7 helicase. By contrast, mammalian initiation sites lack such consensus sequences, and the mammalian ORC does not exhibit sequence specificity. Studies performed over the past decades have identified replication initiation sites in the mammalian genome using sequencing-based assays, raising the question of whether replication initiation occurs at confined sites or in broad zones across the genome. Although recent reports have shown that the licensed MCMs in mammalian cells are broadly distributed, suggesting that ORC-dependent licensing may not determine the initiation sites/zones, they are predominantly located upstream of actively transcribed genes. This review compares the mechanism of replication initiation in yeast and mammalian cells, summarizes the sequencing-based technologies used for the identification of initiation sites/zones, and proposes a possible mechanism of initiation-site/zone selection in mammalian cells. Future directions and challenges in this field are also discussed., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45-MCM-GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome., (© 2024. The Author(s).)

This case report elucidates a scenario involving two sibling sisters born out of consanguineous marriage-one initially presenting with lower respiratory infection, concurrently exhibiting short stature and primary amenorrhoea. Investigation into the primary amenorrhoea unveiled hypergonadotropic hypogonadism, confirmed by the absence of ovaries and a hypoplastic uterus on pelvic MRI. Genetic analysis via whole exome sequencing identified a homozygous variant NM&#95;001282717.2: c.808C>T in the MCM8 gene, located on exon 8 of chromosome 20, inherited in an autosomal recessive manner. The scarcity of primary ovarian insufficiency cases linked to MCM8 highlights the necessity of thoroughly investigating the genetic and clinical consequences of such variants., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Germline-specific genes are usually activated in cancer cells and drive cancer progression; such genes are called cancer-germline or cancer-testis genes. The RNA-binding protein DAZL is predominantly expressed in germ cells and plays a role in gametogenesis as a translational activator or repressor. However, its expression and role in non-small cell lung cancer (NSCLC) are unknown. Here, mining of RNA-sequencing data from public resources and immunohistochemical analysis of tissue microarrays showed that DAZL was expressed exclusively in testis among normal human tissues but ectopically expressed in NSCLC tissues. Testis and NSCLC cells expressed the shorter and longer transcript variants of the DAZL gene, respectively. Overexpression of the longer DAZL transcript promoted tumor growth in a mouse xenograft model. Silencing of DAZL suppressed cell proliferation, colony formation, migration, invasion, and cisplatin resistance in vitro and tumor growth in vivo. Quantitative proteomic analysis based on tandem mass tag and Western blot analysis showed that DAZL upregulated the expression of JAK2 and MCM8. RNA-binding protein immunoprecipitation assays showed that DAZL bound to the mRNA of JAK2 and MCM8. The JAK2 inhibitor fedratinib attenuated the oncogenic outcomes induced by DAZL overexpression, whereas silencing MCM8 counteracted the effects of DAZL overexpression on cisplatin-damaged DNA synthesis and half-maximal inhibitory concentration of cisplatin. In conclusion, DAZL was identified as a novel cancer-germline gene that enhances the translation of JAK2 and MCM8 to promote NSCLC progression and resistance to cisplatin, respectively. These findings suggest that DAZL is a potential therapeutic target in NSCLC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Gastric cancer (GC) is the fourth leading cause of cancer-related death worldwide. Minichromsome maintenance proteins family member 8 (MCM8) assists DNA repair and DNA replication. MCM8 exerts tumor promotor function in multiple digestive system tumors. MCM8 is also considered as a potential cancer therapeutic target., Methods: Bioinformatics methods were used to analyze MCM8 expression and clinicopathological significance. MCM8 expression was detected by immunohistochemistry (IHC) staining and qRT-PCR. MCM8 functions in GC cell were explored by Celigo cell counting, colony formation, wound-healing, transwell, and annexin V-APC staining assays. The target of MCM8 was determined by human gene expression profile microarray. Human phospho-kinase array kit evaluated changes in key proteins after ribosomal protein S15A (RPS15A) knockdown. MCM8 functions were reassessed in xenograft mouse model. IHC detected related proteins expression in mouse tumor sections., Results: MCM8 was significantly upregulated and predicted poor prognosis in GC. High expression of MCM8 was positively correlated with lymph node positive (p < 0.001), grade (p < 0.05), AJCC Stage (p < 0.001), pathologic T (p < 0.01), and pathologic N (p < 0.001). MCM8 knockdown inhibited proliferation, migration, and invasion while promoting apoptosis. RPS15A expression decreased significantly after MCM8 knockdown. It was also the only candidate target, which ranked among the top 10 downregulated differentially expressed genes (DEGs) in sh-MCM8 group. RPS15A was identified as the target of MCM8 in GC. MCM8/RPS15A promoted phosphorylation of P38α, LYN, and p70S6K. Moreover, MCM8 knockdown inhibited tumor growth, RPS15A expression, and phosphorylation of P38α, LYN, and p70S6K in vivo., Conclusions: MCM8 is an oncogene and predicts poor prognosis in GC. MCM8/RPS15A facilitates GC progression., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells., (© 2024. The Author(s).)

MCM8 is a helicase, which participates in DNA replication and tumorigenesis and is upregulated in many human cancers, including lung cancer (LC); however, the function of MCM8 in LC tumour progression is unclear. In this study, we found that MCM8 was expressed at high levels in LC cells and tissues. Further, MCM8 upregulation was associated with advanced tumour grade and lymph node metastasis, and indicated poor prognosis. Silencing of MCM8 suppressed cell growth and migration in vitro and in vivo, while ectopic MCM8 expression promoted cell cycle progression, as well as cell migration, proliferation, and apoptosis. Mechanistically, DNAJC10 was identified as a downstream target of MCM8, using gene array and CO-IP assays. DNAJC10 overexpression combatted the inhibitory activity of MCM8 knockdown on LC progression, while silencing DNAJC10 alleviated the oncogenic function of MCM8 overexpression. MCM8 expression was positively correlated with that of DNAJC10 in LC samples from The Cancer Genome Atlas database, and DNAJC10 upregulation was also associated with poor overall survival of patients with LC. This study indicated that MCM8/DNAJC10 axis plays an important role in in LC development, and maybe as a new potential therapeutic target or a diagnostic biomarker for treating patients with LC., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yáñez-Vilches et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The minichromosome maintenance (MCM) protein is a component of an active helicase that is essential for the initiation of DNA replication. Dysregulation of MCM functions contribute to abnormal cell proliferation and genomic instability. The interactions of MCM with cellular factors, including Cdc45 and GINS, determine the formation of active helicase and functioning of helicase. The functioning of MCM determines the fate of DNA replication and, thus, genomic integrity. This complex is upregulated in precancerous cells and can act as an important tool for diagnostic applications. The MCM protein complex can be an important broad-spectrum therapeutic target in various cancers. Investigations have supported the potential and applications of MCM in cancer diagnosis and its therapeutics. In this article, we discuss the physiological roles of MCM and its associated factors in DNA replication and cancer pathogenesis., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA., (© 2024. The Author(s).)

Malignant tumors constitute a significant category of diseases posing a severe threat to human survival and health, thereby representing one of the most challenging and pressing issues in the field of biomedical research. Due to their malignant nature, which is characterized by a high potential for metastasis, rapid dissemination, and frequent recurrence, the prevailing approach in clinical oncology involves a comprehensive treatment strategy that combines surgery with radiotherapy, chemotherapy, targeted drug therapies, and other interventions. Treatment resistance remains a major obstacle in the comprehensive management of tumors, serving as a primary cause for the failure of integrated tumor therapies and a critical factor contributing to patient relapse and mortality. The Minichromosome Maintenance (MCM) protein family comprises functional proteins closely associated with the development of resistance in tumor therapy.The influence of MCMs manifests through various pathways, encompassing modulation of DNA replication, cell cycle regulation, and DNA damage repair mechanisms. Consequently, this leads to an enhanced tolerance of tumor cells to chemotherapy, targeted drugs, and radiation. Consequently, this review explores the specific roles of the MCM family in various cancer treatment strategies. Its objective is to enhance our comprehension of resistance mechanisms in tumor therapy, thereby presenting novel targets for clinical research aimed at overcoming resistance in cancer treatment. This bears substantial clinical relevance., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bin Zhang reports financial support was provided by the National Natural Science Foundation of China. Bin Zhang reports a relationship with Hunan Provincial University Reform and Development Funding that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Head and neck squamous cell carcinoma (HNSC) is a common malignant tumor in the world and has a poor prognosis. The family of minichromosome maintenance proteins (MCM) improves the stability of genome replication by inhibiting the rate of DNA replication in eukaryotic cells, thus, small changes in physiological MCM levels would increase the instability of gene replication and increase the incidence of tumor formation, most of which are significantly elevated in multiple cancers. However, the expression of different MCM families in HNSC and their prognostic value remain unclear., Methods: ONCOMINE and GEPIA databases were used to analyze the expression of MCMs in HNSC. The Kaplan-Meier plotter database was used to identify molecules with prognostic values. We collected 77 HNSC tissues and 50 normal tissues to validate the results of the bioinformatics analysis by immunohistochemical staining., Results: The expression of MCM3, MCM5 and MCM6 in mRNA and protein levels were higher in HNSC. Moreover, the increased expression of MCM3, MCM5 and MCM6 in mRNA and protein levels predicted better prognosis of HNSC patients. Furthermore, multivariate analysis showed that high expressions of MCM3, MCM5 and MCM6 in protein level may be independent prognostic factors for HNSC patients., Conclusion: The results of this study indicated that MCM3, MCM5 and MCM6 play an important role in occurrence and development in HNSC and might be risk factors for the survival of HNSC patients., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.

Background: A high expression pattern of minichromosome maintenance 2 (MCM2) has been observed in various cancers. MCM2 is a protein involved in the cell cycle and plays a role in cancer growth and differentiation by binding to six members of the MCM subfamily. The MCM protein family includes MCM2 through MCM7., Methods: MCM2 has shown high expression in both lung cancer stem cells (LCSCs) and glioma stem cells (GSCs). We investigated the characteristics of CSCs and the regulation of the epithelial-to-mesenchymal transition (EMT) phenomenon in LCSCs and GSCs by MCM2. Additionally, we explored secreted factors regulated by MCM2., Results: There was a significant difference in survival rates between lung cancer patients and brain cancer patients based on MCM2 expression. MCM2 was found to regulate both markers and regulatory proteins in LCSCs. Moreover, MCM2 is thought to be involved in cancer metastasis by regulating cell migration and invasion, not limited to lung cancer but also identified in glioma. Among chemokines, chemokine (C-X-C motif) ligand 1 (CXCL1) was found to be regulated by MCM2., Conclusions: MCM2 not only participates in the cell cycle but also affects cancer cell growth by regulating the external microenvironment to create a favorable environment for cells. MCM2 is highly expressed in malignant carcinomas, including CSCs, and contributes to the malignancy of various cancers. Therefore, MCM2 may represent a crucial target for cancer therapeutics., (© 2024. The Author(s).)

Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies., (© 2024. The Author(s).)

The replication of DNA requires specialized and intricate machinery. This machinery is known as a replisome and is highly evolutionarily conserved, from simple unicellular organisms such as yeast to human cells. The replisome comprises multiple protein complexes responsible for various steps in the replication process. One crucial component of the replisome is the Cdc45-MCM-GINS (CMG) helicase complex, which unwinds double-stranded DNA and coordinates the assembly and function of other replisome components, including DNA polymerases. The genes encoding the CMG helicase components are essential for initiating DNA replication. In this study, we aimed to investigate how the absence of one copy of the CMG complex genes in heterozygous Saccharomyces cerevisiae cells impacts the cells' physiology and aging. Our data revealed that these cells exhibited a significant reduction in transcript levels for the respective CMG helicase complex proteins, as well as disruptions in the cell cycle, extended doubling times, and alterations in their biochemical profile. Notably, this study provided the first demonstration that cells heterozygous for genes encoding subunits of the CMG helicase exhibited a significantly increased reproductive potential and delayed chronological aging. Additionally, we observed a noteworthy correlation between RNA and polysaccharide levels in yeast and their reproductive potential, as well as a correlation between fatty acid levels and cell doubling times. Our findings also shed new light on the potential utility of yeast in investigating potential therapeutic targets for cancer treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome., (© 2023. Springer Nature Limited.)

Microchromosome maintenance (MCM) proteins are a number of nuclear proteins with significant roles in the development of cancer by influencing the process of cellular DNA replication. Of the MCM protein family, MCM10 is a crucial member that maintains the stability and extension of DNA replication forks during DNA replication and is significantly overexpressed in a variety of cancer tissues, regulating the biological behaviour of cancer cells. But little is understood about MCM10's functional role and regulatory mechanisms in a range of malignancies. We investigate the impact of MCM10 in human cancers by analyzing data from databases like the Gene Expression Profiling Interaction Analysis (GEPIA2), Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA), among others. Possible relationships between MCM10 and clinical staging, diagnosis, prognosis, Mutation burden (TMB), microsatellite instability (MSI), immunological checkpoints, DNA methylation, and tumor stemness were identified. The findings demonstrated that MCM10 expression was elevated in the majority of cancer types and was connected to tumor dryness, immunocytic infiltration, immunological checkpoints, TMB and MSI. Functional enrichment analysis in multiple tumors also identified possible pathways of MCM10 involvement in tumorigenesis. We also discovered promising MCM10-targeting chemotherapeutic drugs. In conclusion, MCM10 may be a desirable pan-cancer biomarker and offer fresh perspectives on cancer therapy., (© 2023. Springer Nature Limited.)

Background: Epigenetic modifications, such as transcription, DNA repair, and replication significantly influence tumour development. Aberrant gene expression and modifications can have a crucial impact on the initiation and progression of tumours. The minichromosome maintenance (MCM) protein family, which is responsible for DNA synthesis, plays a crucial role in tumorigenesis and chemotherapy resistance by regulating the cell cycle and DNA replication stress. Recent studies have shown that dysregulation of the MCMs can lead to these negative outcomes. This study aimed to examine the role of the MCM proteins in DNA synthesis in 33 types of cancers., Methods: Various public databases were used to examine the expression, methylation regulation, mutations, and functions of eight MCM proteins (MCM2-9) in pan-cancer. The study investigated the correlation between abnormal MCM expression and clinical outcomes, including prognosis and drug response. The microRNA-mRNA network upstream of the MCM genes and the downstream signalling pathways were extensively investigated to determine the molecular mechanisms that drive tumour development., Results: The study found that the MCM gene expressions differed depending on the type of cancer; high MCM gene expression was linked to poor overall survival in most cancers. Additionally, MCM gene expression was associated with various immunological features and drug sensitivity. These findings offer important insights for the development of targeted cancer therapies., Conclusions: Altogether, this study reveals that the MCM genes are differentially expressed across various cancers and are associated with clinical prognoses. These genes may influence the occurrence and development of tumours through several pathways, including the PI3K-AKT, PAS/MAPK and TSC/mTOR signalling pathways and immune-related pathways., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.

CMG (Cdc45-MCM-GINS) helicase assembly at the replication origin is the culmination of eukaryotic DNA replication initiation. This process can be reconstructed in vitro using defined factors in Saccharomyces cerevisiae; however, in vertebrates, origin-dependent CMG formation has not yet been achieved partly due to the lack of a complete set of known initiator proteins. Since a microcephaly gene product, DONSON, was reported to remodel the CMG helicase under replication stress, we analyzed its role in DNA replication using a Xenopus cell-free system. We found that DONSON was essential for the replisome assembly. In vertebrates, DONSON physically interacted with GINS and Polε via its conserved N-terminal PGY and NPF motifs, and the DONSON-GINS interaction contributed to the replisome assembly. DONSON's chromatin association during replication initiation required the pre-replicative complex, TopBP1, and kinase activities of S-CDK and DDK. Both S-CDK and DDK required DONSON to trigger replication initiation. Moreover, human DONSON could substitute for the Xenopus protein in a cell-free system. These findings indicate that vertebrate DONSON is a novel initiator protein essential for CMG helicase assembly., (© 2023 The Authors.)

Lung cancer is the most common cause of cancer-related death worldwide, accounting for 1.8 million deaths annually. Analysis of The Cancer Genome Atlas data showed that all members of the minichromosome maintenance (MCM) family (hexamers involved in DNA replication: MCM2-MCM7) were upregulated in lung adenocarcinoma (LUAD) tissues. High expression of MCM4 (P = 0.0032), MCM5 (P = 0.0032), and MCM7 (P = 0.0110) significantly predicted 5-year survival rates in patients with LUAD. Simurosertib (TAK-931) significantly suppressed the proliferation of LUAD cells by inhibiting cell division cycle 7-mediated MCM2 phosphorylation. This finding suggested that MCM2 might be a therapeutic target for LUAD. Moreover, analysis of the epigenetic regulation of MCM2 showed that miR-139-3p, miR-378a-5p, and miR-2110 modulated MCM2 expression in LUAD cells. In patients with LUAD, understanding the role of these miRNAs may improve prognoses., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

With limited therapeutic options for hepatocellular carcinoma (HCC), it is of great significance to investigate the underlying mechanisms and identifying tumor drivers. MCM6, a member of minichromosome maintenance proteins (MCMs), was significantly elevated in HCC progression and associated with poor prognosis. Knockdown of MCM6 significantly inhibited the proliferation and migration of HCC cells with the increased apoptosis ratio and cell cycle arrest, whereas overexpression of MCM6 induced adverse effects. Mechanistically, MCM6 could decrease the P53 activity by inducing the degradation of P53 protein. In addition, MCM6 enhanced the ubiquitination of P53 by recruiting UBE3A to form a triple complex. Furthermore, overexpression of UBE3A significantly rescued the P53 activation and suppression of malignant behaviors mediated by MCM6 inhibition. In conclusion, MCM6 facilitated aggressive phenotypes of HCC cells by UBE3A/P53 signaling, providing potential biomarkers and targets for HCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mitophagy is a major quality control pathway that removes unwanted or dysfunctional mitochondria and plays an essential role in vascular health. Here we show that MCM8 expression is significantly decreased in children with Kawasaki disease (KD) who developed coronary artery aneurysms. Mechanistically, we discovered that nitric oxide signaling promotes TRIM21-mediated MCM8 ubiquitination, which disrupts its interaction with MCM9 and promotes its cytosolic export. In the cytosol, MCM8 relocates to the mitochondria pore-forming proteins and promotes their ubiquitination by TRIM21. In addition, MCM8 directly recruits LC3 via its LC3-interacting region (LIR) motif and initiates mitophagy. This suppresses mitochondrial DNA-mediated activation of type I interferon via cGAS and STING. Mice that are deficient in Mcm8, Trim21 and Nos2 or reconstituted with the East-Asian-specific MCM8-P276 variant develop more severe coronary artery vasculopathy in the Lactobacillus casei extract-induced KD model. Collectively, the data suggest that MCM8 protects vascular health in the KD setting., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The replicative Cdc45-MCM-GINS (CMG) helicase is a large protein complex that functions in the DNA melting and unwinding steps as a component of replisomes during DNA replication in mammalian cells. Although the CMG performs this important role in cell growth, the CMG is not a simple bystander in cell cycle events. Components of the CMG, specifically the MCM precursors, are also involved in maintaining genomic stability by regulating DNA replication fork speeds, facilitating recovery from replicative stresses, and preventing consequential DNA damage. Given these important functions, MCM/CMG complexes are highly regulated by growth factors such as TGF-ß1 and by signaling factors such as Myc, Cyclin E, and the retinoblastoma protein. Mismanagement of MCM/CMG complexes when these signaling mediators are deregulated, and in the absence of the tumor suppressor protein p53, leads to increased genomic instability and is a contributor to tumorigenic transformation and tumor heterogeneity. The goal of this review is to provide insight into the mechanisms and dynamics by which the CMG is regulated during its assembly and activation in mammalian genomes, and how errors in CMG regulation due to oncogenic changes promote tumorigenesis. Finally, and most importantly, we highlight the emerging understanding of the CMG helicase as an exploitable vulnerability and novel target for therapeutic intervention in cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

DNA replication initiation in eukaryotes is tightly regulated through two cell-cycle specific processes, replication licensing to install inactive minichromosome maintenance (MCM) double-hexamers (DH) on origins in early G1 phase and origin firing to assemble and activate Cdc45-Mcm2-7-GINS (CMG) helicases upon S phase entry. Two kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are responsible for driving the association of replication factors with the MCM-DH to form CMG helicases for origin melting and DNA unwinding and eventually replisomes for bi-directional DNA synthesis. In recent years, cryo-electron microscopy studies have generated a collection of structural snapshots for the stepwise assembly and remodeling of the replication initiation machineries, creating a framework for understanding the regulation of this fundamental process at a molecular level. Very recent progress is the structural characterization of the elusive MCM-DH-DDK complex, which provides insights into mechanisms of kinase activation, substrate recognition and selection, as well as molecular role of DDK-mediated MCM-DH phosphorylation in helicase activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: Identify protein contact points between TP53 and minichromosome maintenance (MCM) complex proteins 2, 3, and 5 with high resolution allowing for potential novel Cancer drug design., Methods: A next-generation sequencing-based protein-protein interaction method developed in our laboratory called AVA-Seq was applied to a gold-standard human protein interaction set. Proteins including TP53, MCM2, MCM3, MCM5, HSP90AA1, PCNA, NOD1, and others were sheared and ligated into the AVA-Seq system. Protein-protein interactions were then identified in both mild and stringent selective conditions., Results: Known interactions among MCM2, MCM3, and MCM5 were identified with the AVA-Seq system. The interacting regions detected between these three proteins overlap with the structural data of the MCM complex, and novel domains were identified with high resolution determined by multiple overlapping fragments. Fragments of wild type TP53 were shown to interact with MCM2, MCM3, and MCM5, and details on the location of the interactions were provided. Finally, a mini-network of known and novel cancer protein interactions was provided, which could have implications for fundamental changes in multiple cancers., Conclusion: We provide a high-resolution mini-interactome that could direct novel drug targets and implicate possible effects of specific cancer mutations., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: WASHC1 is a member of the Wiskott-Aldrich syndrome protein (WASP) family and is involved in endosomal protein sorting and trafficking through the generation of filamentous actin (F-actin) via activation of the Arp2/3 complex. There is increasing evidence that WASHC1 is present in the nucleus and nuclear WASHC1 plays important roles in regulating gene transcription, DNA repair as well as maintaining nuclear organization. However, the multi-faceted functions of nuclear WASHC1 still need to be clarified., Methods and Results: We show here that WASHC1 interacts with several components of the minichromosome maintenance (MCM) 2-7 complex by using co-immunoprecipitation and in situ proximity ligation assay. WASHC1-depleted cells display normal DNA replication and S-phase progression. However, loss of WASHC1 sensitizes HeLa cells to DNA replication inhibitor hydroxyurea (HU) and increases chromosome instability of HeLa and 3T3 cells under condition of HU-induced replication stress. Re-expression of nuclear WASHC1 in WASHC1 KO 3T3 cells rescues the deficiency of WASHC1 KO cells in the chromosomal stability after HU treatment. Moreover, chromatin immunoprecipitation assay indicates that WASHC1 associates with DNA replication origins, and knockdown of WASHC1 inhibits MCM protein loading at origins., Conclusions: Since efficient loading of excess MCM2-7 complexes is required for cells to survive replicative stress, these results demonstrate that WASHC1 promotes cell survival and maintain chromosomal stability under replication stress through recruitment of excess MCM complex to origins., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The minichromosome maintenance (MCM) 8/9 helicase is a AAA + complex involved in DNA replication-associated repair. Despite high sequence homology to the MCM2-7 helicase, a precise cellular role for MCM8/9 has remained elusive. We have interrogated the DNA synthesis ability and replication fork stability in cells lacking MCM8 or 9 and find that there is a functional partitioning of MCM8/9 activity between promoting replication fork progression and protecting persistently stalled forks. The helicase function of MCM8/9 aids in normal replication fork progression, but upon persistent stalling, MCM8/9 directs additional downstream stabilizers, including BRCA1 and Rad51, to protect forks from excessive degradation. Loss of MCM8 or 9 slows the overall replication rate and allows for excessive nascent strand degradation, detectable by increased markers of genomic damage. This evidence defines multifunctional roles for MCM8/9 in promoting normal replication fork progression and genome integrity following stress., (© 2022. The Author(s).)

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The MCM2-7 complex is a highly conserved hetero-hexameric protein complex, critical for DNA unwinding at the replicative fork during DNA replication. Overexpression or mutation in MCM2-7 genes is linked to and may drive several cancer types in humans. In mice, mutations in MCM2-7 genes result in growth retardation and mortality. All six MCM2-7 genes are also expressed in the developing mouse CNS, but their role in the CNS is not clear. Here, we use the central nervous system (CNS) of Drosophila melanogaster to begin addressing the role of the MCM complex during development, focusing on the specification of a well-studied neuropeptide expressing neuron: the Tv4/FMRFa neuron. In a search for genes involved in the specification of the Tv4/FMRFa neuron we identified Mcm5 and find that it plays a highly specific role in the specification of the Tv4/FMRFa neuron. We find that other components of the MCM2-7 complex phenocopies Mcm5, indicating that the role of Mcm5 in neuronal subtype specification involves the MCM2-7 complex. Surprisingly, we find no evidence of reduced progenitor proliferation, and instead find that Mcm5 is required for the expression of the type I BMP receptor Tkv, which is critical for the FMRFa expression. These results suggest that the MCM2-7 complex may play roles during CNS development outside of its well-established role during DNA replication., Competing Interests: The authors have declared that no competing interests exist.

Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements., Competing Interests: The authors have declared that no competing interests exist.

Mcm2-7 is the catalytic core of the eukaryotic replicative helicase, which together with CDC45 and the GINS complex unwind parental DNA to generate templates for DNA polymerase. Being a highly regulated and complex enzyme that operates via an incompletely understood multi-step mechanism, molecular probes of Mcm2-7 that interrogate specific mechanistic steps would be useful tools for research and potential future chemotherapy. Based upon a synthetic lethal approach, we previously developed a budding yeast multivariate cell-based high throughput screening (HTS) assay to identify putative Mcm inhibitors by their ability to specifically cause a growth defect in an mcm mutant relative to a wild-type strain[1]. Here, as proof of concept, we used this assay to screen a 1280-member compound library (LOPAC) for potential Mcm2-7 inhibitors. Primary screening and dose-dependent retesting identified twelve compounds from this library that specifically inhibited the growth of the Mcm mutant relative to the corresponding wild-type strain (0.9 % hit rate). Secondary assays were employed to rule out non-specific DNA damaging agents, establish direct protein-ligand interaction via biophysical methods, and verify in vivo DNA replication inhibition via fluorescence activated cell sorter analysis (FACS). We identified one agent (β-carboline-3-carboxylic acid N-methylamide, CMA) that physically bound to the purified Mcm2-7 complex (Kd app 119 µM), and at slightly higher concentrations specifically blocked S-phase cell cycle progression of the wild-type strain. In total, identification of Mcm2-7 as a CMA target validates our synthetic lethal HTS assay paradigm as a tool to identify chemical probes for the Mcm2-7 replicative helicase., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

The eukaryotic CDC45/MCM2-7/GINS (CMG) helicase unwinds the DNA double helix during DNA replication. The GINS subcomplex is required for helicase activity and is, therefore, essential for DNA replication and cell viability. Here, we report the identification of 7 individuals from 5 unrelated families presenting with a Meier-Gorlin syndrome-like (MGS-like) phenotype associated with hypomorphic variants of GINS3, a gene not previously associated with this syndrome. We found that MGS-associated GINS3 variants affecting aspartic acid 24 (D24) compromised cell proliferation and caused accumulation of cells in S phase. These variants shortened the protein half-life, altered key protein interactions at the replisome, and negatively influenced DNA replication fork progression. Yeast expressing MGS-associated variants of PSF3 (the yeast GINS3 ortholog) also displayed impaired growth, S phase progression defects, and decreased Psf3 protein stability. We further showed that mouse embryos homozygous for a D24 variant presented intrauterine growth retardation and did not survive to birth, and that fibroblasts derived from these embryos displayed accelerated cellular senescence. Taken together, our findings implicate GINS3 in the pathogenesis of MGS and support the notion that hypomorphic variants identified in this gene impaired cell and organismal growth by compromising DNA replication.

Minichromosome maintenance (Mcm) proteins are well-known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS-nonspecific group consisting of Mcm13, Mcm16-19, and Mcm21-22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high-fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non-overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure-function of the ARS-nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms., (© 2021 Wiley Periodicals LLC.)

The replication machinery that synthesizes new copies of chromosomal DNA is located at the junction where double-stranded DNA is separated into its two strands. This replication fork DNA structure is at the heart of most assays involving DNA helicases. The helicase enzyme unwinds the replication fork structure into two single-stranded templates which are converted into two daughter duplexes by other proteins, including DNA polymerases. In eukaryotes, the CMG (Cdc45/Mcm2-7/GINS) helicase plays the pivotal role of unwinding the parental duplex DNA and at the same time interacts with numerous other proteins, including the leading strand polymerase, Pol ɛ. This chapter first describes how we design and prepare synthetic replication forks used in our CMG-related assays. Then we describe how to load CMG onto the fork. The Mcm2-7 motor subunits of CMG form a closed ring, as do all cellular replicative helicases, that encircles ssDNA for helicase function. Thus, the first step in these assays is the loading of CMG onto the fork DNA, followed by DNA unwinding and replication. We explain protocols for different strategies of preloading CMG onto the DNA fork using different ATP analogues. Additionally, the presence of Mcm10, an intimate partner of CMG, affects how CMG is preloaded onto a fork substrate., (Copyright © 2022 Elsevier Inc. All rights reserved.)

YTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in acute myeloid leukemia (AML) and that it is required for the proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We found that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem and progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides compelling evidence that shows an oncogenic role and a distinct mechanism of YTHDC1 in AML., (© 2021 by The American Society of Hematology.)

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., (© 2021 The Author(s).)

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin-recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases., Competing Interests: SG, LF, JG, SB No competing interests declared, (© 2021, Gupta et al.)

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.

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response., (© 2021. The Author(s).)

MCM8/9 is a complex involved in homologous recombination (HR) repair pathway. MCM8/9 dysfunction can cause genome instability and result in primary ovarian insufficiency (POI). However, the mechanism underlying these effects is largely unknown. Here, we report crystal structures of the N-terminal domains (NTDs) of MCM8 and MCM9, and build a ring-shaped NTD structure based on a 6.6 Å resolution cryoelectron microscopy map. This shows that the MCM8/9 complex forms a 3:3 heterohexamer in an alternating pattern. A positively charged DNA binding channel and a putative ssDNA exit pathway for fork DNA unwinding are revealed. Based on the atomic model, the potential effects of the clinical POI mutants are interpreted. Surprisingly, the zinc-finger motifs are found to be capable of binding an iron atom as well. Overall, our results provide a model for the formation of the MCM8/9 complex and provide a path for further studies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)