Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

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.

Sister chromatid cohesion establishment during DNA replication termination.

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.

Quantity and quality of minichromosome maintenance protein complexes couple replication licensing to genome integrity.

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.

TopBP1 utilises a bipartite GINS binding mode to support genome replication.

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

Disorders in the CMG helicase complex increase the proliferative capacity and delay chronological aging of budding yeast.

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.

RIF1 regulates early replication timing in murine B cells.

The mammalian DNA replication timing (RT) program is crucial for the proper functioning and integrity of the genome. The best-known mechanism for controlling RT is the suppression of late origins of replication in heterochromatin by RIF1. Here, we report that in antigen-activated, hypermutating murine B lymphocytes, RIF1 binds predominantly to early-replicating active chromatin and promotes early replication, but plays a minor role in regulating replication origin activity, gene expression and genome organization in B cells. Furthermore, we find that RIF1 functions in a complementary and non-epistatic manner with minichromosome maintenance (MCM) proteins to establish early RT signatures genome-wide and, specifically, to ensure the early replication of highly transcribed genes. These findings reveal additional layers of regulation within the B cell RT program, driven by the coordinated activity of RIF1 and MCM proteins.

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

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

The structural mechanism of dimeric DONSON in replicative helicase activation.

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.

A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint.

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.

DONSON is required for CMG helicase assembly in the mammalian cell cycle.

DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier-Gorlin syndrome. The other 12 encode components of the CDC45-MCM-GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45-MCM-GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45-MCM-GINS during S-phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45-MCM-GINS helicase from S-phase cells and our data indicate that DONSON is dispensable for loading of the MCM2-7 helicase core onto chromatin during G1-phase, but instead is essential for CDC45-MCM-GINS assembly during S-phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier-Gorlin syndrome.

Pan-Cancer Multi-Omics Analysis of Minichromosome Maintenance Proteins (MCMs) Expression in Human Cancers.

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.

MCM4 acts as a biomarker for LUAD prognosis.

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

DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation.

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Our data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.

In silico protein interaction screening uncovers DONSON's role in replication initiation.

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.

DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in Caenorhabditis elegans.

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.

Structural and mechanistic insights into the MCM8/9 helicase complex.

MCM8 and MCM9 form a functional helicase complex (MCM8/9) that plays an essential role in DNA homologous recombination repair for DNA double-strand break. However, the structural characterization of MCM8/9 for DNA binding/unwinding remains unclear. Here, we report structures of the MCM8/9 complex using cryo-electron microscopy single particle analysis. The structures reveal that MCM8/9 is arranged into a heterohexamer through a threefold symmetry axis, creating a central channel that accommodates DNA. Multiple characteristic hairpins from the N-terminal oligosaccharide/oligonucleotide (OB) domains of MCM8/9 protrude into the central channel and serve to unwind the duplex DNA. When activated by HROB, the structure of MCM8/9's N-tier ring converts its symmetry from C3 to C1 with a conformational change that expands the MCM8/9's trimer interface. Moreover, our structural dynamic analyses revealed that the flexible C-tier ring exhibited rotary motions relative to the N-tier ring, which is required for the unwinding ability of MCM8/9. In summary, our structural and biochemistry study provides a basis for understanding the DNA unwinding mechanism of MCM8/9 helicase in homologous recombination.

Minichromosome maintenance protein family member 6 mediates hepatocellular carcinoma progression by recruiting UBE3A to induce P53 ubiquitination.

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.

Changing protein-DNA interactions promote ORC binding-site exchange during replication origin licensing.

During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC (origin recognition complex) can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA-binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally controlled DNA sliding of helicase-loading intermediates and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA-binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely oriented Mcm2-7 helicases to ensure bidirectional DNA replication.

Novel role of DONSON in CMG helicase assembly during vertebrate DNA replication initiation.

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.

Minichromosome maintenance proteins in lung adenocarcinoma: Clinical significance and therapeutic targets.

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.