Search

The eukaryotic helicase MCM2-7, is loaded by ORC, Cdc6 and Cdt1 as a double-hexamer onto replication origins. The insertion of DNA into the helicase leads to partial MCM2-7 ring closure, while ATP hydrolysis is essential for consecutive steps in pre-replicative complex (pre-RC) assembly. Currently it is unknown how MCM2-7 ring closure and ATP-hydrolysis are controlled. A cryo-EM structure of an ORC-Cdc6-Cdt1-MCM2-7 intermediate shows a remodelled, fully-closed Mcm2/Mcm5 interface. The Mcm5 C-terminus (C5) contacts Orc3 and specifically recognises this closed ring. Interestingly, we found that normal helicase loading triggers Mcm4 ATP-hydrolysis, which in turn leads to reorganisation of the MCM2-7 complex and Cdt1 release. However, defective MCM2-7 ring closure, due to mutations at the Mcm2/Mcm5 interface, leads to MCM2-7 ring splitting and complex disassembly. As such we identify Mcm4 as the key ATPase in regulating pre-RC formation. Crucially, a stable Mcm2/Mcm5 interface is essential for productive ATP-hydrolysis-dependent remodelling of the helicase., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The ability to initiate DNA replication is a critical step in the proliferation of all organisms. In bacteria, this process is mediated by an ATP-dependent replication initiator protein, DnaA, which recognizes and melts replication origin (oriC) elements. Despite decades of biochemical and structural work, a mechanistic understanding of how DnaA recognizes and unwinds oriC has remained enigmatic. A recent study by Pelliciari et al. provides important new structural insights into how DnaA from Bacillus subtilis recognizes and processes its cognate oriC, showing how DnaA uses sequence features encoded in the origin to engage melted DNA. Comparison of the DnaA-oriC structure with archaeal/eukaryl replication origin complexes based on Orc-family proteins reveals a high degree of similarity in origin engagement by initiators from di domains of life, despite fundamental differences in origin melting mechanisms. These findings provide valuable insights into bacterial replication initiation and highlight the intriguing evolutionary history of this fundamental biological process., (© 2024 Wiley Periodicals LLC.)

Autonomously replicating sequences (ARSs) are important accessories in episomal vectors that allow them to be replicated and stably maintained within transformants. Despite their importance, no information on ARSs in diatoms has been reported. Therefore, we attempted to identify ARS candidates in the model diatom, Phaeodactylum tricornutum, via chromatin immunoprecipitation sequencing. In this study, subunits of the origin recognition complex (ORC), ORC2 and ORC4, were used to screen for ARS candidates. ORC2 and ORC4 bound to 355 sites on the P. tricornutum genome, of which 69 were constantly screened after multiple attempts. The screened ARS candidates had an AT-richness of approximately 50% (44.39-52.92%) and did not have conserved sequences. In addition, ARS candidates were distributed randomly but had a dense distribution pattern at several sites. Their positions tended to overlap with those of the genetic region (73.91%). Compared to the ARSs of several other eukaryotic organisms, the characteristics of the screened ARS candidates are complex. Thus, our findings suggest that the diatom has a distinct and unique native ARSs., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Exploring novel targets for non-small cell lung cancer (NSCLC) remains of utmost importance. This study focused on ORC6 (origin recognition complex subunit 6), investigating its expression and functional significance within NSCLC. Analysis of the TCGA-lung adenocarcinoma database revealed a notable increase in ORC6 expression in lung adenocarcinoma tissues, correlating with reduced overall survival, advanced disease stages, and other key clinical parameters. Additionally, in patients undergoing surgical resection of NSCLC at a local hospital, ORC6 mRNA and protein levels were elevated in NSCLC tissues while remaining low in adjacent normal tissues. Comprehensive bioinformatics analyses across various cancers suggested that ORC6 might play a significant role in crucial cellular processes, such as mitosis, DNA synthesis and repair, and cell cycle progression. Knocking down ORC6 using virus-delivered shRNA in different NSCLC cells, both primary and immortalized, resulted in a significant hindrance to cell proliferation, cell cycle progression, migration and invasion, accompanied by caspase-apoptosis activation. Similarly, employing CRISPR-sgRNA for ORC6 knockout (KO) exhibited significant anti-NSCLC cell activity. Conversely, increasing ORC6 levels using a viral construct augmented cell proliferation and migration. Silencing or knockout of ORC6 in primary NSCLC cells led to reduced expression of several key cyclins, including Cyclin A2, Cyclin B1, and Cyclin D1, whereas their levels increased in NSCLC cells overexpressing ORC6. In vivo experiments demonstrated that intratumoral injection of ORC6 shRNA adeno-associated virus markedly suppressed the growth of primary NSCLC cell xenografts. Reduced ORC6 levels, downregulated cyclins, and increased apoptosis were evident in ORC6-silenced NSCLC xenograft tissues. In summary, elevated ORC6 expression promotes NSCLC cell growth., (© 2024. The Author(s).)

In eukaryotes, the origin recognition complex (ORC) faciliates the assembly of pre-replicative complex (pre-RC) at origin DNA for replication licensing. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit is crucial for this process. Removing a segment (residues 176-200) from Orc2-IDR or mutating a key isoleucine (194) significantly inhibits replication initiation across the genome. These Orc2-IDR mutants are capable of assembling the ORC-Cdc6-Cdt1-Mcm2-7 intermediate, which exhibits impaired ATP hydrolysis and fails to be convered into the subsequent Mcm2-7-ORC complex and pre-RC. These defects can be partially rescued by the Orc2-IDR peptide. Moreover, the phosphorylation of this Orc2-IDR region by S cyclin-dependent kinase blocks its binding to Mcm2-7 complex, causing a defective pre-RC assembly. Our findings provide important insights into the multifaceted roles of ORC in supporting origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase., (© 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).)

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.)

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).)

The human immunodeficiency virus type 1 (HIV-1) reservoir consists of latently infected cells which present a major obstacle to achieving a functional cure for HIV-1. The formation and maintenance of HIV-1 latency have been extensively studied, and latency-reversing agents (LRAs) that can reactivate latent HIV-1 by targeting the involved host factors are developed; however, their clinical efficacies remain unsatisfactory. Therefore, it is imperative to identify novel targets for more potential candidates or better combinations for LRAs. In this study, we utilized CRISPR affinity purification in situ of regulatory elements system to screen for host factors associated with the HIV-1 long terminal repeat region that could potentially be involved in HIV-1 latency. We successfully identified that origin recognition complex 1 (ORC1), the largest subunit of the origin recognition complex, contributes to HIV-1 latency in addition to its function in DNA replication initiation. Notably, ORC1 is enriched on the HIV-1 promoter and recruits a series of repressive epigenetic elements, including DNMT1 and HDAC1/2, and histone modifiers, such as H3K9me3 and H3K27me3, thereby facilitating the establishment and maintenance of HIV-1 latency. Moreover, the reactivation of latent HIV-1 through ORC1 depletion has been confirmed across various latency cell models and primary CD4 + T cells from people living with HIV-1. Additionally, we comprehensively validated the properties of liquid-liquid phase separation (LLPS) of ORC1 from multiple perspectives and identified the key regions that promote the formation of LLPS. This property is important for the recruitment of ORC1 to the HIV-1 promoter. Collectively, these findings highlight ORC1 as a potential novel target implicated in HIV-1 latency and position it as a promising candidate for the development of novel LRAs., Importance: Identifying host factors involved in maintaining human immunodeficiency virus type 1 (HIV-1) latency and understanding their mechanisms prepares the groundwork to discover novel targets for HIV-1 latent infection and provides further options for the selection of latency-reversing agents in the "shock" strategy. In this study, we identified a novel role of the DNA replication factor origin recognition complex 1 (ORC1) in maintaining repressive chromatin structures surrounding the HIV-1 promoter region, thereby contributing to HIV-1 latency. This discovery expands our understanding of the non-replicative functions of the ORC complex and provides a potential therapeutic strategy for HIV-1 cure., Competing Interests: The authors declare no conflict of interest.

Lipopolysaccharide (LPS)-activated pro-inflammatory responses play a critical role in sepsis, a life-threatening condition. This study investigates the role of origin recognition complex subunit 6 (ORC6) in LPS responses in macrophages and monocytes. Silencing ORC6 using targeted shRNA significantly reduced LPS-induced expression and production of IL-1β (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IL-6 (interleukin-6) in THP-1 human macrophages, peripheral blood mononuclear cells (PBMCs), and bone marrow-derived macrophages (BMDMs). Additionally, ORC6 knockout (KO) via the CRISPR/Cas9 method in THP-1 macrophages inhibited LPS-induced pro-inflammatory responses, while ectopic overexpression of ORC6 enhanced LPS-induced expression and production of pro-inflammatory cytokines. ORC6 is crucial for the activation of the nuclear factor kappa B (NFκB) signaling cascade in macrophages and monocytes. LPS-induced NFκB activation was largely inhibited by ORC6 silencing or KO, but potentiated following ORC6 overexpression. Mechanistically, ORC6 associated with nuclear p65 after LPS stimulation, an interaction necessary for NFκB activation. Overexpression of ORC6 did not recover the reduced pro-inflammatory response to LPS in THP-1 macrophages with silenced p65. Furthermore, the NFκB inhibitor BMS-345,541 nearly eliminated the pro-inflammatory response enhanced by ORC6 overexpression in response to LPS. Further studies revealed that ORC6 depletion inhibited NFκB activation induced by double-stranded RNA (dsRNA) and high mobility group box 1 (HMGB1) in THP-1 macrophages. In vivo experiments demonstrated that macrophage-specific knockdown of ORC6 protected mice from LPS-induced septic shock and inhibited LPS-stimulated production of IL-1β, TNF-α, and IL-6 in mouse serum. ORC6 silencing also inhibited LPS-induced NFκB activation in ex vivo cultured PBMCs following macrophage-specific knockdown of ORC6. These findings highlight ORC6 as a pivotal mediator in LPS-induced NFκB activation and the pro-inflammatory response in sepsis, suggesting that targeting ORC6 could be a novel therapeutic strategy for managing sepsis and related inflammatory conditions., (© 2024. The Author(s).)

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hoggard 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 discovery of novel oncotargets for glioma is of immense significance. We here explored the expression patterns, biological functions, and underlying mechanisms associated with ORC6 (origin recognition complex 6) in glioma. Through the bioinformatics analyses, we found a significant increase in ORC6 expression within human glioma tissues, correlating with poorer overall survival, higher tumor grade, and wild-type isocitrate dehydrogenase status. Additionally, ORC6 overexpression is detected in glioma tissues obtained from locally-treated patients and across various primary/established glioma cells. Further bioinformatics scrutiny revealed that genes co-expressed with ORC6 are enriched in multiple signaling cascades linked to cancer. In primary and immortalized (A172) glioma cells, depleting ORC6 using specific shRNA or Cas9-sgRNA knockout (KO) significantly decreased cell viability and proliferation, disrupted cell cycle progression and mobility, and triggered apoptosis. Conversely, enhancing ORC6 expression via a lentiviral construct augmented malignant behaviors in human glioma cells. ORC6 emerged as a crucial regulator for the expression of key oncogenic genes, including Cyclin A2, Cyclin B2, and DNA topoisomerase II (TOP2A), within glioma cells. Silencing or KO of ORC6 reduced the mRNA and protein levels of these genes, while overexpression of ORC6 increased their expression in primary glioma cells. Bioinformatics analyses further identified RBPJ as a potential transcription factor of ORC6. RBPJ shRNA decreased ORC6 expression in primary glioma cells, while its overexpression increased it. Additionally, significantly enhanced binding between the RBPJ protein and the proposed ORC6 promoter region was detected in glioma tissues and cells. In vivo experiments demonstrated a significant reduction in the growth of patient-derived glioma xenografts in the mouse brain subsequent to ORC6 KO. ORC6 depletion, inhibited proliferation, decreased expression of Cyclin A2/B2/TOP2A, and increased apoptosis were detected within these ORC6 KO intracranial glioma xenografts. Altogether, RBPJ-driven ORC6 overexpression promotes glioma cell growth, underscoring its significance as a promising therapeutic target., (© 2024. The Author(s).)

In previous studies, we purified the DUB-module of the Drosophila SAGA complex and showed that a number of zinc proteins interact with it, including Aef1 and CG10543. In this work, we conducted a genome-wide study of the Aef1 and CG10543 proteins and showed that they are localized predominantly on the promoters of active genes. The binding sites of these proteins co-localize with the SAGA and dSWI/SNF chromatin modification and remodeling complexes, as well as with the ORC replication complex. It has been shown that the Aef1 and CG10543 proteins are involved in the regulation of the expression of some genes on the promoters of which they are located. Thus, the Aef1 and CG10543 proteins are new participants in the cell transcriptional network and co-localize with the main transcription and replication complexes of Drosophila.

Prostate cancer (PCa) is the most prevalent malignant tumor of the genitourinary system, and metastatic disease has a significant impact on the prognosis of PCa patients. As a result, knowing the processes of PCa development can help patients achieve better outcomes. Here, we investigated the expression and function of ORC6 in PCa. Our findings indicated that ORC6 was elevated in advanced PCa tissues. Patients with PCa who exhibited high levels of ORC6 had a poor prognosis. Following that, we investigated the function of ORC6 in PCa progression using a variety of functional experiments both in vivo and in vitro, and discovered that ORC6 knockdown inhibited PCa cell proliferation, growth, and migration. Furthermore, RNA-seq was employed to examine the molecular mechanism of PCa progression. The results revealed that ORC6 might promote the expression of PLK1, a serine/threonine kinase in PCa cells. We also discovered that ORC6 as a novel miR-361-5p substrate using database analysis, and miR-361-5p was found to lower ORC6 expression. Additionally, RNA immunoprecipitation (RIP) and luciferase reporter tests revealed that the transcription factor E2F1 could regulate ORC6 expression in PCa cells. PLK1 overexpression or miR-361-5p inhibitor treatment effectively removed the inhibitory effects caused by ORC6 silencing. Notably, our data showed that therapeutically targeting the miR-361-5p/ORC6/PLK1 axis may be a viable therapy option for PCa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: a non-repetitive motif whose binding requires Orc1-BP4 and Orc4-IH, and a repetitive one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The first step toward eukaryotic genome duplication is loading of the replicative helicase onto chromatin. This 'licensing' step initiates with the recruitment of the origin recognition complex (ORC) to chromatin, which is thought to occur via ORC's ATP-dependent DNA binding and encirclement activity. However, we have previously shown that ATP binding is dispensable for the chromatin recruitment of fly ORC, raising the question of how metazoan ORC binds chromosomes. We show here that the intrinsically disordered region (IDR) of fly Orc1 is both necessary and sufficient for recruitment of ORC to chromosomes in vivo and demonstrate that this is regulated by IDR phosphorylation. Consistently, we find that the IDR confers the ORC holocomplex with ATP-independent DNA binding activity in vitro. Using phylogenetic analysis, we make the surprising observation that metazoan Orc1 IDRs have diverged so markedly that they are unrecognizable as orthologs and yet we find that these compositionally homologous sequences are functionally conserved. Altogether, these data suggest that chromatin is recalcitrant to ORC's ATP-dependent DNA binding activity, necessitating IDR-dependent chromatin tethering, which we propose poises ORC to opportunistically encircle nucleosome-free regions as they become available., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Based on experimentally determined average inter-origin distances of ~100 kb, DNA replication initiates from ~50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the origin recognition complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and five ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ~7.5 million union origins identified by all datasets, only 0.27% (20,250 shared origins) were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques, suggesting extensive variability in origin usage and identification. Also, 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF-binding sites, G-quadruplex sites, and activating histone marks, these overlaps are comparable or less than that of known transcription start sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ~13,000 reproducible ORC-binding sites in human cancer cells, and only 4.5% were within 1 kb of the ~11,000 union MCM2-7-binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, Saccharomyces cerevisiae . Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites., Competing Interests: MT, ZW, ZS, ES, YS, AD, CZ No competing interests declared, (© 2023, Tian et al.)

Glioma is one of the most common and difficult to cure malignant primary tumors of the central nervous system. Long non-coding RNA (lncRNA) has been reported to play important functions in biological processes of many tumors, including glioma. In our study, we aimed to reveal the role and molecular mechanisms of lncRNA COX10-AS1 in regulating the progression of glioma. First of all, we showed that lncRNA COX10-AS1 was significantly increased in glioma tissues and cell lines, and high-expressed COX10-AS1 was associated with a poor prognosis in glioma patients. Moreover, through performing the functional experiments, including CCK-8, colony formation and Transwell assays, we confirmed that COX10-AS1 ablation curbed cell proliferation, migration and invasion in glioblastoma (GBM) cells. In addition, we uncovered that there existed a regulatory relationship that COX10-AS1 upregulated OCR6 by sponging miR-1-3p in GBM cells, and the following rescue assays demonstrated that both miR-1-3p downregulation and origin recognition complex subunit 6 (ORC6) overexpression rescued cell viability, migration and invasion in the COX10-AS1-deficient GBM cells. Consistently, we also verified that COX10-AS1 promoted tumorigenesis of the GBM cells in vivo through modulating the miR-1-3p/ORC6 axis. On the whole, our findings indicated a novel ceRNA pattern in which COX10-AS1 elevated OCR6 expression via sponging miR-1-3p, therefore boosting tumorigenesis in glioma, and we firstly discussed the underlying mechanisms by which the COX10-AS1/miR-1-3p/ORC6 axis affected the progression of glioma., (Copyright © 2023 IBRO. Published by Elsevier Inc. All rights reserved.)

Guanine (G)-rich nucleic acid sequences can form diverse G-quadruplex structures located in functionally significant genome regions, exerting regulatory control over essential biological processes, including DNA replication in vivo. During the initiation of DNA replication, Cdc6 is recruited by the origin recognition complex (ORC) to target specific chromosomal DNA sequences. This study reveals that human Cdc6 interacts with G-quadruplex structure through a distinct region within the N-terminal intrinsically disordered region (IDR), encompassing residues 7-20. The binding region assumes a hook-type conformation, as elucidated by the NMR solution structure in complex with htel21T 18 . Significantly, mutagenesis and in vivo investigations confirm the highly specific nature of Cdc6's recognition of G-quadruplex. This research enhances our understanding of the fundamental mechanism governing the interaction between G-quadruplex and the N-terminal IDR region of Cdc6, shedding light on the intricate regulation of DNA replication processes., Competing Interests: Declaration of competing interest The authors declare that we 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.)

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 ( Orc6 ), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence. To explore the potential function of Orc6 in spermatogonia, the C18-4 cell line was transfected with control or Orc6 siRNA. Subsequently, 5-ethynyl-2-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, flow cytometry, and western blot were used to evaluate its effects on proliferation and apoptosis. It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells. Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated (Wnt)/β-catenin signaling. Western blot revealed that the expression of β-catenin protein and its phosphorylation (Ser675) were significantly decreased when silencing the expression of ORC6. Our findings indicated that Orc6 was upregulated in spermatogonia, whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling., (Copyright © 2023 Copyright: © The Author(s)(2023).)

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.)

Objective: To analyze the genetic characteristics of a child with Meier-Gorlin syndrome (MGS) due to a homozygous variant of the ORC6 gene., Methods: A child who was admitted to the Children's Hospital Affiliated to Soochow University on March 25, 2019 due to growth retardation was selected as the study subject. Clinical data of the child was collected. Whole exome sequencing was carried out for the child. Candidate variant was validated by Sanger sequencing and bioinformatic analysis., Results: The child, a 8-year-and-3-month-old male, has featured short stature, small ears, bilateral cryptorchidism and patellar dysplasia. His parents were of first cousins. The child was found to harbor a homozygous c.712A>T (p.K238*) missense variant of the ORC6 gene, which may lead to premature termination of protein translation. Sanger sequencing confirmed that both of his parents were heterozygous carriers. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was classified as pathogenic (PVS1_Moderate+PM2_Supporting+PM3+PP3+PP4)., Conclusion: The homozygous c.712A>T (p.K238*) variant probably underlay the MGS in this child.

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., (© 2023 The Authors.)

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.

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation.

The Escherichia coli replication origin oriC contains the initiator ATP-DnaA-Oligomerization Region (DOR) and its flanking duplex unwinding element (DUE). In the Left-DOR subregion, ATP-DnaA forms a pentamer by binding to R1, R5M and three other DnaA boxes. The DNA-bending protein IHF binds sequence-specifically to the interspace between R1 and R5M boxes, promoting DUE unwinding, which is sustained predominantly by binding of R1/R5M-bound DnaAs to the single-stranded DUE (ssDUE). The present study describes DUE unwinding mechanisms promoted by DnaA and IHF-structural homolog HU, a ubiquitous protein in eubacterial species that binds DNA sequence-non-specifically, preferring bent DNA. Similar to IHF, HU promoted DUE unwinding dependent on ssDUE binding of R1/R5M-bound DnaAs. Unlike IHF, HU strictly required R1/R5M-bound DnaAs and interactions between the two DnaAs. Notably, HU site-specifically bound the R1-R5M interspace in a manner stimulated by ATP-DnaA and ssDUE. These findings suggest a model that interactions between the two DnaAs trigger DNA bending within the R1/R5M-interspace and initial DUE unwinding, which promotes site-specific HU binding that stabilizes the overall complex and DUE unwinding. Moreover, HU site-specifically bound the replication origin of the ancestral bacterium Thermotoga maritima depending on the cognate ATP-DnaA. The ssDUE recruitment mechanism could be evolutionarily conserved in eubacteria., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase-the minichromosome maintenance (MCM) complex-at replication origins 1 . Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes 2-4 . However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by orc1 mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Error-free genome duplication and accurate cell division are critical for cell survival. In all three domains of life, bacteria, archaea, and eukaryotes, initiator proteins bind replication origins in an ATP-dependent manner, play critical roles in replisome assembly, and coordinate cell-cycle regulation. We discuss how the eukaryotic initiator, Origin recognition complex (ORC), coordinates different events during the cell cycle. We propose that ORC is the maestro driving the orchestra to coordinately perform the musical pieces of replication, chromatin organization, and repair., (© 2023 The Authors. BioEssays published by Wiley Periodicals LLC.)

Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well defined. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially overlapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efficient deposition of the heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. The distinct roles of the two ORC1 proteins may be a feature common to other organisms with duplicated ORC1 genes and a major difference with animal cells., (© 2023. The Author(s).)

Errors occurring during DNA replication can result in inaccurate replication, incomplete replication, or re-replication, resulting in genome instability that can lead to diseases such as cancer or disorders such as autism. A great deal of progress has been made toward understanding the entire process of DNA replication in eukaryotes, including the mechanism of initiation and its control. This review focuses on the current understanding of how the origin recognition complex (ORC) contributes to determining the location of replication initiation in the multiple chromosomes within eukaryotic cells, as well as methods for mapping the location and temporal patterning of DNA replication. Origin specification and configuration vary substantially between eukaryotic species and in some cases co-evolved with gene-silencing mechanisms. We discuss the possibility that centromeres and origins of DNA replication were originally derived from a common element and later separated during evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: DNA replication alteration is a hallmark of patients with lung adenocarcinoma (LUAD) and is frequently observed in LUAD progression. Origin recognition complex (ORC) 1, ORC2, ORC3, ORC4, ORC5, and ORC6 form a replication-initiator complex to mediate DNA replication, which plays a key role in carcinogenesis, while their roles in LUAD remain poorly understood., Methods: The mRNA and protein expression of ORCs was confirmed by the GEPIA, HPA, CPTAC, and TCGA databases. The protein-protein interaction network was analyzed by the GeneMANIA database. Functional enrichment was confirmed by the Metascape database. The effects of ORCs on immune infiltration were validated by the TIMER database. The prognostic significance of ORCs in LUAD was confirmed by the KM-plot and GENT2 databases. DNA alteration and protein structure were determined in the cBioProtal and PDB databases. Moreover, the protein expression and prognostic value of ORCs were confirmed in our LUAD data sets by immunohistochemistry (IHC) staining., Results: ORC mRNA and protein were significantly increased in patients with LUAD compared with corresponding normal tissue samples. The results of IHC staining analysis were similar result to those of the above bioinformatics analysis. Furthermore, ORC1 and ORC6 had significant prognostic values for LUAD patients. Furthermore, the ORC cooperatively promoted LUAD development by driving DNA replication, cellular senescence, and metabolic processes., Conclusion: The ORC, especially ORC1/6, has important prognostic and expression significance for LUAD patients., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Replication licensing, a prerequisite of DNA replication, helps to ensure once-per-cell-cycle genome duplication. Some DNA replication-initiation proteins are sequentially loaded onto replication origins to form pre-replicative complexes (pre-RCs). ORC and Noc3p bind replication origins throughout the cell cycle, providing a platform for pre-RC assembly. We previously reported that cell cycle-dependent ORC dimerization is essential for the chromatin loading of the symmetric MCM double-hexamers. Here, we used Saccharomyces cerevisiae separation-of-function NOC3 mutants to confirm the separable roles of Noc3p in DNA replication and ribosome biogenesis. We also show that an essential and cell cycle-dependent Noc3p dimerization cycle regulates the ORC dimerization cycle. Noc3p dimerizes at the M-to-G 1 transition and de-dimerizes in S-phase. The Noc3p dimerization cycle coupled with the ORC dimerization cycle enables replication licensing, protects nascent sister replication origins after replication initiation, and prevents re-replication. This study has revealed a new mechanism of replication licensing and elucidated the molecular mechanism of Noc3p as a mediator of ORC dimerization in pre-RC formation., (© 2023 Amin et al.)

The smallest subunit of the human Origin Recognition Complex, hOrc6, is required for DNA replication progression and plays an important role in mismatch repair (MMR) during S-phase. However, the molecular details of how hOrc6 regulates DNA replication and DNA damage response remain to be elucidated. Orc6 levels are elevated upon specific types of genotoxic stress, and it is phosphorylated at Thr229, predominantly during S-phase, in response to oxidative stress. Many repair pathways, including MMR, mediate oxidative DNA damage repair. Defects in MMR are linked to Lynch syndrome, predisposing patients to many cancers, including colorectal cancer. Orc6 levels are known to be elevated in colorectal cancers. Interestingly, tumor cells show reduced hOrc6-Thr229 phosphorylation compared to adjacent normal mucosa. Further, elevated expression of wild-type and the phospho-dead forms of Orc6 results in increased tumorigenicity, implying that in the absence of this "checkpoint" signal, cells proliferate unabated. Based on these results, we propose that DNA-damage-induced hOrc6-pThr229 phosphorylation during S-phase facilitates ATR signaling in the S-phase, halts fork progression, and enables assembly of repair factors to mediate efficient repair to prevent tumorigenesis. Our study provides novel insights into how hOrc6 regulates genome stability.

All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~10 2 -fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we find that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

ORC2 is a small subunit of the origin recognition complex (ORC), which is important for gene replication. The ORC2 WH domain recognizes dsDNA sequences with its flexible β-sheet hairpins as anchors. Here, we report near-complete NMR backbone and side chain resonance assignments of the WH domain and study the backbone relaxation of the WH domain. These studies will contribute to further understanding of the structure-function relationship of the ORC protein., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes., (© 2022. The Author(s).)

Meier−Gorlin syndrome (MGS) is a rare genetic developmental disorder that causes primordial proportional dwarfism, microtia, the absence of or hypoplastic patellae and other skeletal anomalies. Skeletal symptoms overlapping with other syndromes make MGS difficult to diagnose clinically. We describe a 3-year-old boy with short stature, recurrent respiratory infections, short-rib dysplasia, tower head and facial dysmorphisms who was admitted to the Tomsk Genetic Clinic to verify a clinical diagnosis of Jeune syndrome. Clinical exome sequencing revealed two variants (compound heterozygosity) in the ORC6 gene: c.2T>C(p.Met1Thr) and c.449+5G>A. In silico analysis showed the pathogenicity of these two mutations and predicted a decrease in donor splicing site strength for c.449+5G>A. An in vitro minigene assay indicated that variant c.449+5G>A causes complete skipping of exon 4 in the ORC6 gene. The parents requested urgent prenatal testing for MGS for the next pregnancy, but it ended in a miscarriage. Our results may help prevent MGS misdiagnosis in the future. We also performed in silico and functional analyses of ORC6 mutations and developed a restriction fragment length polymorphism and haplotype-based short-tandem-repeat assay for prenatal genetic testing for MGS. These findings should elucidate MGS etiology and improve the quality of genetic counselling for affected families.

As a typical species of microsporidium, Nosema bombycis is the pathogen causing the pébrine disease of silkworm. Rapid proliferation of N. bombycis in host cells requires replication of genetic material. As eukaryotic origin recognition protein, origin recognition complex (ORC) plays an important role in regulating DNA replication, and Orc1 is a key subunit of the origin recognition complex. In this study, we identified the Orc1 in the microsporidian N. bombycis (NbOrc1) for the first time. The NbOrc1 gene contains a complete ORF of 987 bp in length that encodes a 328 amino acid polypeptide. Indirect immunofluorescence results showed that NbOrc1 were colocalized with Nbactin and NbSAS-6 in the nuclei of N. bombycis. Subsequently, we further identified the interaction between the NbOrc1 and Nbactin by CO-IP and Western blot. These results imply that Orc1 may be involved in the proliferation of the microsporidian N. bombycis through interacting with actin., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Unwinding of the replication origin and loading of DNA helicases underlie the initiation of chromosomal replication. In Escherichia coli, the minimal origin oriC contains a duplex unwinding element (DUE) region and three (Left, Middle, and Right) regions that bind the initiator protein DnaA. The Left/Right regions bear a set of DnaA-binding sequences, constituting the Left/Right-DnaA subcomplexes, while the Middle region has a single DnaA-binding site, which stimulates formation of the Left/Right-DnaA subcomplexes. In addition, a DUE-flanking AT-cluster element (TATTAAAAAGAA) is located just outside of the minimal oriC region. The Left-DnaA subcomplex promotes unwinding of the flanking DUE exposing TT[A/G]T(T) sequences that then bind to the Left-DnaA subcomplex, stabilizing the unwound state required for DnaB helicase loading. However, the role of the Right-DnaA subcomplex is largely unclear. Here, we show that DUE unwinding by both the Left/Right-DnaA subcomplexes, but not the Left-DnaA subcomplex only, was stimulated by a DUE-terminal subregion flanking the AT-cluster. Consistently, we found the Right-DnaA subcomplex-bound single-stranded DUE and AT-cluster regions. In addition, the Left/Right-DnaA subcomplexes bound DnaB helicase independently. For only the Left-DnaA subcomplex, we show the AT-cluster was crucial for DnaB loading. The role of unwound DNA binding of the Right-DnaA subcomplex was further supported by in vivo data. Taken together, we propose a model in which the Right-DnaA subcomplex dynamically interacts with the unwound DUE, assisting in DUE unwinding and efficient loading of DnaB helicases, while in the absence of the Right-DnaA subcomplex, the AT-cluster assists in those processes, supporting robustness of replication initiation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest related to the content of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In eukaryotes, the origin recognition complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for prereplication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression after pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the chromatin-association of MutLα, thus enabling efficient MMR. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S phase.

Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Precise DNA replication is pivotal for ensuring the accurate inheritance of genetic information. To avoid genetic instability, each DNA fragment needs to be amplified only once per cell cycle. DNA replication in eukaryotes starts with the binding of the origin recognition complex (ORC) to the origins of DNA replication. The genes encoding ORC subunits have been conserved across eukaryotic evolution and are essential for the initiation of DNA replication. In this study, we conducted an extensive physiological and aging-dependent analysis of heterozygous cells lacking one copy of ORC genes in the BY4743 background. Cells with only one copy of the ORC genes showed a significant decrease in the level of ORC mRNA, a delay in the G1 phase of the cell cycle, and an extended doubling time. Here, we also show that the reducing the levels of Orc1-6 proteins significantly extends both the budding and average chronological lifespans. Heterozygous ORC/orcΔ and wild-type diploid cells easily undergo haploidization during chronological aging. This ploidy shift might be related to nutrient starvation or the inability to survive under stress conditions. A Raman spectroscopy analysis helped us to strengthen the hypothesis of the importance of lipid metabolism and homeostasis in aging.

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.)

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2-ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Epigenetic inheritance of heterochromatin requires DNA-sequence-independent propagation mechanisms, coupling to RNAi, or input from DNA sequence, but how DNA contributes to inheritance is not understood. Here, we identify a DNA element (termed "maintainer") that is sufficient for epigenetic inheritance of pre-existing histone H3 lysine 9 methylation (H3K9me) and heterochromatin in Schizosaccharomyces pombe but cannot establish de novo gene silencing in wild-type cells. This maintainer is a composite DNA element with binding sites for the Atf1/Pcr1 and Deb1 transcription factors and the origin recognition complex (ORC), located within a 130-bp region, and can be converted to a silencer in cells with lower rates of H3K9me turnover, suggesting that it participates in recruiting the H3K9 methyltransferase Clr4/Suv39h. These results suggest that, in the absence of RNAi, histone H3K9me is only heritable when it can collaborate with maintainer-associated DNA-binding proteins that help recruit the enzyme responsible for its epigenetic deposition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In vertebrates, single cell analyses of replication timing patterns brought to light a very well controlled program suggesting a tight regulation on initiation sites. Mapping of replication origins with different methods has revealed discrete preferential sites, enriched in promoters and potential G-quadruplex motifs, which can aggregate into initiation zones spanning several tens of kilobases (kb). Another characteristic of replication origins is a nucleosome-free region (NFR). A modified yeast strain containing a humanized origin recognition complex (ORC) fires new origins at NFRs revealing their regulatory role. In cooperation with NFRs, the histone variant H2A.Z facilitates ORC loading through di-methylation of lysine 20 of histone H4. Recent studies using genome editing methods show that efficient initiation sites associated with transcriptional activity can synergize over several tens of kb by establishing physical contacts and lead to the formation of early domains of DNA replication demonstrating a co-regulation between replication initiation and transcription., (© 2021 Wiley Periodicals LLC.)

The protein stability of the initiation factors Orc2, Orc3, Orc4, and Cdc6 was analyzed after UV light exposure in two human cell lines. In the cell line with higher repair capacity, HEK 293, no changes in the cell cycle distribution or in the protein levels of the investigated factors were detected. In HeLa cells that are characterized by lower repair capacity, UV irradiation caused a reduction of the levels of Cdc6, Orc2 and Orc3, but not of Orc4 or triggered apoptosis. The appearance of the truncated 49 kDa form of Cdc6 suggested the involvement of the caspase pathway in the degradation of the proteins. Reduced protein levels of Cdc6 were detected in UV damaged HeLa cells in which the apoptotic process was blocked with the caspase inhibitor Z-VAD-fmk, indicating that the degradation of Cdc6 is mediated by the proteasome pathway instead. In the presence of caffeine, an inhibitor of the cell cycle checkpoint kinases, Cdc6 was stabilized, demonstrating that its degradation is controlled by the DNA damage cell cycle checkpoint. We conclude that in response to DNA damage, the activation of origins of replication can be prevented by the degradation of Cdc6, most likely through the proteasome pathway., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)