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

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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.

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To initiate DNA replication, it is essential to properly assemble a pair of replicative helicases at each replication origin. While the general principle of this process applies universally from prokaryotes to eukaryotes, the specific mechanisms governing origin selection, helicase loading, and subsequent helicase activation vary significantly across different species. Recent advancements in cryo-electron microscopy (cryo-EM) have revolutionized our ability to visualize large protein or protein-DNA complexes involved in the initiation of DNA replication. Complemented by real-time single-molecule analysis, the available high-resolution cryo-EM structures have greatly enhanced our understanding of the dynamic regulation of this process at origin DNA. This review primarily focuses on the latest structural discoveries that shed light on the key molecular machineries responsible for driving replication initiation, with a particular emphasis on the assembly of pre-replication complex (pre-RC) in eukaryotes.

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RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

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Precise DNA replication is fundamental for genetic inheritance. In eukaryotes, replication initiates at multiple origins that are first "licensed" and subsequently "fired" to activate DNA synthesis. Despite the success in identifying origins with specific DNA motifs in Saccharomyces cerevisiae, no consensus sequence or sequences with a predictive value of replication origins have been recognized in metazoan genomes. Rather, epigenetic rules and chromatin structures are believed to play important roles in governing the selection and activation of replication origins. We propose that replication initiation is facilitated by a group of sequence-specific "replication pioneer factors," which function to increase chromatin accessibility and foster a chromatin environment that is conducive to the loading of the prereplication complex. Dysregulation of the function of these factors may lead to gene duplication, genomic instability, and ultimately the occurrence of pathological conditions such as cancer.

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Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG helicases (Cdc45⋅Mcm2-7⋅GINS) at each origin. This requires several replication firing factors (including TopBP1, RecQL4, and DONSON) whose exact roles are still under debate. How two helicases are correctly assembled and activated at each origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered that replication initiation is surprisingly dynamic. First, TopBP1 transiently binds to the origin and dissociates before the start of DNA synthesis. Second, two Cdc45 are recruited together, even though Cdc45 alone cannot dimerize. Next, two copies of DONSON and two GINS simultaneously arrive at the origin, completing the assembly of two CMG helicases. Finally, RecQL4 is recruited to the CMG⋅DONSON⋅DONSON⋅CMG complex and promotes DONSON dissociation and CMG activation via its ATPase activity.

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There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, 'a site that binds Mcm in G1' might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here, we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least three orders of magnitude. Published data quantifying single-stranded DNA (ssDNA) during S phase revealed replication initiation among the most abundant 1600 of these sites, with replication activity decreasing with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5500 sites. Specifically, these sites: (1) appeared in intergenic nucleosome-free regions flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. We conclude that, if sites at which Mcm double hexamers are loaded can function as replication origins, then DNA replication origins are at least threefold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than widely assumed.

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For over a decade, it has been known that yeast Sld2, Dpb11, GINS and Polε form the pre-loading complex (pre-LC), which is recruited to a CDC45-bound MCM2-7 complex by the Sld3/Sld7 heterodimer in a phospho-dependent manner. Whilst functional orthologs of Dbp11 (TOPBP1), Sld3 (TICRR) and Sld7 (MTBP) have been identified in metazoans, controversy has surrounded the identity of the Sld2 ortholog. It was originally proposed that the RECQ helicase, RECQL4, which is mutated in Rothmund-Thomson syndrome, represented the closest vertebrate ortholog of Sld2 due to a small region of sequence homology at its N-Terminus. However, there is no clear evidence that RECQL4 is required for CMG loading. Recently, new findings suggest that the functional ortholog of Sld2 is actually DONSON, a replication fork stability factor mutated in a range of neurodevelopmental disorders characterised by microcephaly, short stature and limb abnormalities. These studies show that DONSON forms a complex with TOPBP1, GINS and Polε analogous to the pre-LC in yeast, which is required to position the GINS complex on the MCM complex and initiate DNA replication. Taken together with previously published functions for DONSON, these observations indicate that DONSON plays two roles in regulating DNA replication, one in promoting replication initiation and one in stabilising the fork during elongation. Combined, these findings may help to uncover why DONSON mutations are associated with such a wide range of clinical deficits.

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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.

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Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence. Fusion proteins (ELP-poly(d)-POIs), composed of elastin-like polypeptide (ELP) fused with protein of interests (POIs) via poly(aspartic acid) (poly(d)), formed the protein nanoparticles based on the temperature responsivity of ELP sequences, leading to multiply displayed POIs on the protein nanoparticles. In the present study, we focused on porcine circovirus type 2 replication initiation protein (Rep), which covalently conjugated with DNA aptamers, and NanoLuc luciferase (Nluc), which emitted a strong bioluminescence, as POIs. ELP-poly(d)-Rep and ELP-poly(d)-Nluc were constructed and formed the protein nanoparticles with multiply displayed Nluc and Rep (DNA aptamer) that amplified the bioluminescence signal and tumor recognition ability. Mucin-1 (MUC1)-overexpressing human breast tumor MCF7 cells and MUC1-recognizing aptamer (MUC1 aptamer) were selected as models. The MUC1 aptamer-conjugated protein nanoparticles exhibited a 13.7-fold higher bioluminescence signal to MCF-7 cells than to human embryonic kidney 293 (HEK293) cells, which express low levels of MUC1. Furthermore, the protein nanoparticles could detect up to 70.7 cells/mL of MCF-7 cells from a cell suspension containing HEK-293. The protein nanoparticles with multiple Rep and Nluc show a great potential as a material for detecting CTCs.

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DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1-24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. In vitro and in vivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214-223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome.

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Eukaryotic cells use chromatin marks to regulate the initiation of DNA replication. The origin recognition complex (ORC)-associated protein ORCA plays a critical role in heterochromatin replication in mammalian cells by recruiting the initiator ORC, but the underlying mechanisms remain unclear. Here, we report crystal and cryo-electron microscopy structures of ORCA in complex with ORC's Orc2 subunit and nucleosomes, establishing that ORCA orchestrates ternary complex assembly by simultaneously recognizing a highly conserved peptide sequence in Orc2, nucleosomal DNA, and repressive histone trimethylation marks through an aromatic cage. Unexpectedly, binding of ORCA to nucleosomes prevents chromatin array compaction in a manner that relies on H4K20 trimethylation, a histone modification critical for heterochromatin replication. We further show that ORCA is necessary and sufficient to specifically recruit ORC into chromatin condensates marked by H4K20 trimethylation, providing a paradigm for studying replication initiation in specific chromatin contexts. Collectively, our findings support a model in which ORCA not only serves as a platform for ORC recruitment to nucleosomes bearing specific histone marks but also helps establish a local chromatin environment conducive to subsequent MCM2-7 loading.

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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.

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Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different stresses. The small RNA DsrA helps bacteria adapt to environments by binding to multiple targets, but its association with the cell cycle remains unclear. Detection by flow cytometry, we first found that the knockout of dsrA promoted replication initiation, and corresponding overexpression of DsrA inhibited replication initiation in Escherichia coli. The absence of the chaperone protein Hfq, the DNA replication negative regulator protein Dps, or the transcription factor OxyR, was found to cause DsrA to no longer inhibit replication initiation. Excess DsrA promotes expression of the oxyR and dps gene, whereas ß-galactosidase activity assay showed that deleting oxyR limited the enhancement of dps promoter transcriptional activity by DsrA. OxyR is a known positive regulator of Dps. Our data suggests that the effect of DsrA on replication initiation requires Hfq and that the upregulation of Dps expression by OxyR in response to DsrA levels may be a potential regulatory pathway for the negative regulation of DNA replication initiation.

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Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

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In metazoan cells, DNA replication initiates from thousands of genomic loci scattered throughout the genome called DNA replication origins. Origins are strongly associated with euchromatin, particularly open genomic regions such as promoters and enhancers. However, over a third of transcriptionally silent genes are associated with DNA replication initiation. Most of these genes are bound and repressed by the Polycomb repressive complex-2 (PRC2) through the repressive H3K27me3 mark. This is the strongest overlap observed for a chromatin regulator with replication origin activity. Here, we asked whether Polycomb-mediated gene repression is functionally involved in recruiting DNA replication origins to transcriptionally silent genes. We show that the absence of EZH2, the catalytic subunit of PRC2, results in increased DNA replication initiation, specifically in the vicinity of EZH2 binding sites. The increase in DNA replication initiation does not correlate with transcriptional de-repression or the acquisition of activating histone marks but does correlate with loss of H3K27me3 from bivalent promoters.

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In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.

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In the conventional model of transcriptional activation, transcription factors bind to response elements and recruit co-factors, including histone acetyltransferases. Contrary to this model, we show that the histone acetyltransferase KAT7 (HBO1/MYST2) is required genome wide for histone H3 lysine 14 acetylation (H3K14ac). Examining neural stem cells, we find that KAT7 and H3K14ac are present not only at transcribed genes but also at inactive genes, intergenic regions, and in heterochromatin. KAT7 and H3K14ac were not required for the continued transcription of genes that were actively transcribed at the time of loss of KAT7 but indispensable for the activation of repressed genes. The absence of KAT7 abrogates neural stem cell plasticity, diverse differentiation pathways, and cerebral cortex development. Re-expression of KAT7 restored stem cell developmental potential. Overexpression of KAT7 enhanced neuron and oligodendrocyte differentiation. Our data suggest that KAT7 prepares chromatin for transcriptional activation and is a prerequisite for gene activation.

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Plasmids found in Acinetobacter species contribute to the spread of antibiotic resistance genes. They appear to be largely confined to this genus and cannot be typed with available tools and databases. Here, a method for distinguishing and typing these plasmids was developed using a curated, non-redundant set of 621 complete sequences of plasmids from Acinetobacter baumannii. Plasmids were separated into 3 groups based on the Pfam domains of the encoded replication initiation (Rep) protein and a fourth group that lack an identifiable Rep protein. The rep genes of each Rep-encoding group (n = 13 Rep_1, n = 107 RepPriCT_1, n = 351 Rep_3) were then clustered using a threshold of >95% nucleotide identity to define 80 distinct types. Five Rep_1 subgroups, designated R1_T1 to R1-T5, were identified and a sixth reported recently was added. Each R1 type corresponded to a conserved small plasmid sequence. The RepPriCT_1 plasmids fell into 5 subgroups, designated RP-T1 to RP-T5 and the Rep_3 plasmids comprised 69 distinct types (R3-T1 to R3-T69). Three R1, 2 RP and 32 R3 types are represented by only a single plasmid. Over half of the plasmids belong to the 4 most abundant types: the RP-T1 plasmids (n = 97), which include conjugation genes and are often associated with various acquired antibiotic resistance genes, and R3-T1, R3-T2 and R3-T3 (n = 95, 30 and 45, respectively). To facilitate typing and the identification of plasmids in draft genomes using this framework, we established the Acinetobacter Typing database containing representative nucleotide and protein sequences of the type markers (https://github.com/MehradHamidian/AcinetobacterPlasmidTyping). IMPORTANCE Though they contribute to the dissemination of genes that confer resistance to clinically important carbapenem and aminoglycoside antibiotics used to treat life-threatening Acinetobacter baumannii infections, plasmids found in Acinetobacter species have not been well studied. As these plasmids do not resemble those found in other Gram-negative pathogens, available typing systems are unsuitable. The plasmid typing system developed for A. baumannii plasmids with an identifiable rep gene will facilitate the classification and tracking of sequenced plasmids. It will also enable the detection of plasmid-derived contigs present in draft genomes that are widely ignored currently. Hence, it will assist in the tracking of resistance genes and other genes that affect survival in the environment, as they spread through the population. As identical or similar plasmids have been found in other Acinetobacter species, the typing system will also be broadly applicable in identifying plasmids in other members of the genus.

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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.

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