CMG helicase disassembly is essential and driven by two pathways in budding yeast.

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 SCFDia2. 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.

TRAIP is a master regulator of DNA interstrand crosslink repair.

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.

CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination.

A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its Mcm7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are enriched on chromatin when CMG unloading is blocked. This approach identified the E3 ubiquitin ligase CRL2Lrr1, a specific p97 complex, other potential regulators of termination, and many replisome components. We show that Mcm7 ubiquitylation and CRL2Lrr1 binding to chromatin are temporally linked and occur only during replication termination. In the absence of CRL2Lrr1, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large subcomplex of the vertebrate replisome that includes DNA Pol ε is retained on DNA. Our data identify CRL2Lrr1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.

Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing.

The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1*Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.

The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase.

Complex cellular processes are driven by the regulated assembly and disassembly of large multiprotein complexes. While we are beginning to understand the molecular mechanism for assembly of the eukaryotic DNA replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Recently, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases have now been shown to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here, using Xenopus laevis egg extract cell-free system and biochemical approaches, we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly during S-phase. We show only Ubxn7, however, facilitates efficient replisome disassembly. Ubxn7 delivers this role through its interaction via independent domains with both Cul2Lrr1 and p97 to allow coupling between Mcm7 ubiquitylation and its removal from chromatin. Our data therefore characterize Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates and a rationale for the efficacy of the Cul2Lrr1 replisome unloading pathway in unperturbed S-phase.

Pharmacoinformatics and molecular dynamics simulation-based phytochemical screening of neem plant (Azadiractha indica) against human cancer by targeting MCM7 protein.

Minichromosome maintenance complex component 7 (MCM7) belongs to the minichromosome maintenance family that is important for the initiation of eukaryotic DNA replication. Overexpression of the MCM7 protein is relative to cellular proliferation and responsible for aggressive malignancy in various cancers. Mechanistically, inhibition of MCM7 significantly reduces the cellular proliferation associated with cancer. To date, no effective small molecular candidate has been identified that can block the progression of cancer induced by the MCM7 protein. Therefore, the study has been designed to identify small molecular-like natural drug candidates against aggressive malignancy associated with various cancers by targeting MCM7 protein. To identify potential compounds against the targeted protein a comprehensive in silico drug design including molecular docking, ADME (Absorption, Distribution, Metabolism and Excretion), toxicity, and molecular dynamics (MD) simulation approaches has been applied. Seventy phytochemicals isolated from the neem tree (Azadiractha indica) were retrieved and screened against MCM7 protein by using the molecular docking simulation method, where the top four compounds have been chosen for further evaluation based on their binding affinities. Analysis of ADME and toxicity properties reveals the efficacy and safety of the selected four compounds. To validate the stability of the protein-ligand complex structure MD simulations approach has also been performed to the protein-ligand complex structure, which confirmed the stability of the selected three compounds including CAS ID:105377-74-0, CID:12308716 and CID:10505484 to the binding site of the protein. In the study, a comprehensive data screening process has performed based on the docking, ADMET properties, and MD simulation approaches, which found a good value of the selected four compounds against the targeted MCM7 protein and indicates as a promising and effective human anticancer agent.

CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.

Homozygous mutation in MCM7 causes autosomal recessive primary microcephaly and intellectual disability.

BACKGROUND: Minichromosomal maintenance (MCM) complex components 2, 4, 5 and 6 have been linked to human disease with phenotypes including microcephaly and intellectual disability. The MCM complex has DNA helicase activity and is thereby important for the initiation and elongation of the replication fork and highly expressed in proliferating neural stem cells. METHODS: Whole-exome sequencing was applied to identify the genetic cause underlying the neurodevelopmental disease of the index family. The expression pattern of Mcm7 was characterised by performing quantitative real-time PCR, in situ hybridisation and immunostaining. To prove the disease-causative nature of identified MCM7, a proof-of-principle experiment was performed. RESULTS: We reported that the homozygous missense variant c.793G>A/p.A265T (g.7:99695841C>T, NM_005916.4) in MCM7 was associated with autosomal recessive primary microcephaly (MCPH), severe intellectual disability and behavioural abnormalities in a consanguineous pedigree with three affected individuals. We found concordance between the spatiotemporal expression pattern of Mcm7 in mice and a proliferative state: Mcm7 expression was higher in early mouse developmental stages and in proliferative zones of the brain. Accordingly, Mcm7/MCM7 levels were detectable particularly in undifferentiated mouse embryonal stem cells and human induced pluripotent stem cells compared with differentiated neurons. We further demonstrate that the downregulation of Mcm7 in mouse neuroblastoma cells reduces cell viability and proliferation, and, as a proof-of-concept, that this is counterbalanced by the overexpression of wild-type but not mutant MCM7. CONCLUSION: We report mutations of MCM7 as a novel cause of autosomal recessive MCPH and intellectual disability and highlight the crucial function of MCM7 in nervous system development.

Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.

When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.

Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint.

Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

NCOA4 transcriptional coactivator inhibits activation of DNA replication origins.

NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.

Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes.

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

MCM7 affects the cisplatin resistance of liver cancer cells and the development of liver cancer by regulating the PI3K/Akt signaling pathway.

OBJECTIVE: Aberrant DNA replication is regarded as a component of cancer development. Minichromosome maintenance protein 7 (MCM7), which is critical for the initiation of DNA replication, is overexpressed in multiple malignancies. The effect of MCM7 on cell proliferation, apoptosis, and drug resistance of liver cancer and its mechanism were investigated in this study. METHODS: MCM7 expression in normal liver cells, liver cancer cell lines, and tissues, as well as adjacent tissues, was determined by qRT-PCR. CCK-8 and flow cytometry was performed to detect cell viability, apoptosis, and cell cycle, respectively. The related mRNA and protein expressions were detected by qRT-PCR and western blot. RESULTS: High expression of MCM7 was found in liver cancer tissues and cells, which results in notably lower survival time of patients. Cisplatin (DDP) could inhibit cell proliferation and affect MCM7 expression. Silencing of MCM7 inhibited cell viability, promoted cell apoptosis, arrested cell cycle at G1 phase, and enhanced the effect of DDP on cancer cells, while overexpression of MCM7 did the opposite. Moreover, silencing of MCM7 inhibited cyclinD1 and Ki-67 expressions. The overexpression of MCM7 increased phosphorylation levels of PI3K and AKT, activated the PI3K/AKT pathway, and weakened the inhibitory effect of DDP on the PI3K/AKT pathway. CONCLUSION: Silencing of MCM7 may inhibit cell proliferation and promote apoptosis by regulating the PI3K/AKT pathway to affect the cell cycle, thus affecting the development of liver cancer, and improving the sensitivity of liver cancer cells to DDP.

PRMT5 promotes colorectal cancer growth by interaction with MCM7.

Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse arginine methylation of histones and non-histone proteins. Accumulating evidence indicates that PRMT5 promotes cancer development and progression. However, its function in colorectal cancer (CRC) is poorly understood. In this study, we revealed the oncogenic roles of PRMT5 in CRC. We found that PRMT5 promoted CRC cell proliferation, migration and invasion in vitro and in vivo. We identified minichromosome maintenance-7 (MCM7) as the direct PRMT5-binding partner. A co-immunoprecipitation (co-IP) assay indicated that PRMT5 physically interacted with MCM7 and that the direct binding domain was located between residues 1-248 in MCM7. In addition, our results from analysis of 99 CRC tissues and 77 adjacent non-cancerous tissues indicated that the PRMT5 and MCM7 expression levels were significantly higher in CRC tissues than in control tissues, which was further confirmed by bioinformatic analysis using TCGA and GEO datasets. We also found that MCM7 promoted CRC cell proliferation, migration and invasion in vitro. Furthermore, we observed that increased PRMT5 expression predicted unfavourable patient survival in CRC patients and in the subgroup of patients with a tumour size of ≤5 cm. These data suggested that PRMT5 and MCM7 might be novel potential targets for the treatment of CRC.

The Immunohistochemical Expression of MCM-3, -5, and -7 Proteins in the Uterine Fibroids.

Uterine fibroids are the most common mesenchymal uterine neoplasms; their prevalence is estimated in 40%-60% of women under 35 and in 70%-80% of women over 50 years of age. The current research aims to focus on the etiopathogenesis of uterine fibroids, the factors that affect their growth, and markers with diagnostic and prognostic properties. The MCM (minichromosome maintenance) protein family consists of peptides whose primary function is participation in the molecular mechanism of creating replication forks while regulating DNA synthesis. The aim of this work was to determine the proliferative potential of uterine fibroid cells based on the expression of the Ki-67 antigen and the MCMs-i.e., MCM-3, MCM-5, and MCM-7. In addition, the expression of estrogen (ER) and progesterone (PgR) receptors was evaluated and correlated with the expression of the abovementioned observations. Ultimately, received results were analyzed in terms of clinical and pathological data. MATERIALS AND METHODS: In forty-four cases of uterine fibroids, immunohistochemical reactions were performed. A tissue microarray (TMA) technique was utilized and analyzed cases were assessed in triplicate. Immunohistochemistry was performed using antibodies against Ki-67 antigen, ER, PgR, MCM-3, MCM-5, and MCM-8 on an automated staining platform. Reactions were digitalized by a histologic scanner and quantified utilizing dedicated software for nuclear analysis. Assessment was based on quantification expression of the three histiospots, each representing one case in TMA. RESULTS: In the study group (uterine fibroids), statistically significant stronger expression of all the investigated MCMs was observed, as compared to the control group. In addition, moderate and strong positive correlations were found between all tested proliferative markers. The expression of the MCM-7 protein also correlated positively with ER and PgR. With regard to clinical and pathological data, there was a negative correlation between the expression of MCMs and the number of both pregnancies and births. Significant reductions in MCM-5 and MCM-7 expression were observed in the group of women receiving oral hormonal contraceptives, while smoking women showed an increase in MCM-7, ER, and PgR. CONCLUSIONS: Uterine fibroid cells have greater proliferative potential, as evaluated by expression of the Ki-67 antigen and MCMs, than unaltered myometrial cells of the uterine corpus. The expression of MCM-7 was found to have strong or moderate correlations in all assessed relations. In the context of the clinical data, as well evident proliferative potential of MCMs, further studies are strongly recommended.

FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

Resting cells rely on the DNA helicase component MCM2 to build cilia.

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.

Computational Screening of Natural Compounds for Identification of Potential Anti-Cancer Agents Targeting MCM7 Protein.

Minichromosome maintenance complex component 7 (MCM7) is involved in replicative licensing and the synthesis of DNA, and its overexpression is a fascinating biomarker for various cancer types. There is currently no effective agent that can prevent the development of cancer caused by the MCM7 protein. However, on the molecular level, inhibiting MCM7 lowers cancer-related cellular growth. With this purpose, this study screened 452 biogenic compounds extracted from the UEFS Natural Products dataset against MCM protein by using the in silico art of technique. The hit compounds UEFS99, UEFS137, and UEFS428 showed good binding with the MCM7 protein with binding energy values of -9.95, -8.92, and -8.71 kcal/mol, which was comparatively higher than that of the control compound ciprofloxacin (-6.50). The hit (UEFS99) with the minimum binding energy was picked for molecular dynamics (MD) simulation investigation, and it demonstrated stability at 30 ns. Computational prediction of physicochemical property evaluation revealed that these hits are non-toxic and have good drug-likeness features. It is suggested that hit compounds UEFS99, UEFS137, and UEFS428 pave the way for further bench work validation in novel inhibitor development against MCM7 to fight the cancers.

MCM7 silencing promotes cutaneous melanoma cell autophagy and apoptosis by inactivating the AKT1/mTOR signaling pathway.

Cutaneous melanoma (CM) has become a major public health concern. Studies illustrate that minichromosome maintenance protein 7 (MCM7) participate in various diseases including skin disease. Our study aimed to study the effects of MCM7 silencing on CM cell autophagy and apoptosis by modulating the AKT threonine kinase 1 (AKT1)/mechanistic target of rapamycin kinase (mTOR) signaling pathway. Initially, microarray analysis was used to screen the CM-related gene expression data as well as differentially expressed genes. Subsequently, MCM7 expression vector and lentivirus RNA used for MCM7 silencing (LV-shRNA-MCM7) were constructed, and these vectors, dimethyl sulfoxide (DMSO) and AKT activator SC79 were then introduced into CM cell line SK-MEL-2 to validate the role of MCM7 in cell autophagy, viability, apoptosis, cell cycle, migration, and invasion. To further investigate the regulatory mechanisms of MCM7 in CM progress, the expression of MCM7, AKT1, mTOR, cyclin D1, as well as autophagy and apoptosis relative factors, such as LC3B, SOD2, DJ-1, p62, Bcl-2, Bax, and caspase-3 in melanoma cells was determined. MCM7 might mediate the AKT1/mTOR signaling pathway to influence the progress of melanoma. MCM7 silencing contributed to the increased expression of Bax, capase-3, and autophagy-related genes (LC3B, SOD2, and DJ-1), but decreased the expression of Bcl-2, which suggested that MCM7 silencing promoted autophagy and cell apoptosis. At the same time, MCM7 silencing also attenuated cell viability, invasion, and migration, and reduced the cyclin D1 expression and protein levels of p-AKT1 and p-mTOR. Taken together, MCM7 silencing inhibited CM via inactivation of the AKT1/mTOR signaling pathway.

Mechanisms of eukaryotic replisome disassembly.

DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.