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Replication is initiated bidirectionally in the three domains of life by the assembly of two replication forks at an origin of replication. This is made possible by the recruitment of two replicative helicases to a nucleoprotein platform built at the origin of replication with the initiator protein. The reason why replication is initiated bidirectionally has never been experimentally addressed due to the lack of a suitable biological system. Using genetic and genomic approaches, we show that upon depletion of DciA, replication is no longer initiated bidirectionally at the origin of replication of Vibrio cholerae chromosome 1. We show that following unidirectional replication on the left replichore, nascent DNA strands at ori1 anneal to each other to form a double-stranded DNA end. While this DNA end can be efficiently resected in recB+ cells, only a few cells use it to trigger replication on the right replichore. In most DciA-depleted cells, chromosome 1 is degraded leading to cell death. Our results suggest that DciA is essential to ensuring bidirectional initiation of replication in bacteria, preventing a cascade of deleterious events following unidirectional replication initiation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chromosomal DNA replication is a fundamental process of life, involving the assembly of complex machinery and dynamic regulation. In this study, we reconstructed a bacterial replication module (pRC) by artificially clustering 23 genes involved in DNA replication and sequentially deleting these genes from their naturally scattered loci on the chromosome of Escherichia coli. The integration of pRC into the chromosome, moving from positions farther away to close to the replication origin, leads to an enhanced efficiency in DNA synthesis, varying from lower to higher. Strains containing replication modules exhibited increased DNA replication by accelerating the replication fork movement and initiating chromosomal replication earlier in the replication cycle. The minimized module pRC16, containing only replisome and elongation encoding genes, exhibited chromosomal DNA replication efficiency comparable to that of pRC. The replication module demonstrated robust and rapid DNA replication, regardless of growth conditions. Moreover, the replication module is plug-and-play, and integrating it into Mb-sized extrachromosomal plasmids improves their genetic stability. Our findings indicate that DNA replication, being a fundamental life process, can be artificially reconstructed into replication functional modules. This suggests potential applications in DNA replication and the construction of synthetic modular genomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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).)

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., (© 2024 Wiley Periodicals LLC.)

G-quadruplex (G4) structures are found in eukaryotic cell replication origins, but their role in origin function remains unclear. In this study G4 motifs are found in the lytic DNA replication origin (oriLyt) of human cytomegalovirus (HCMV) and recombinant viruses show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and biochemical/biophysical analyses show that G4 formation in ER-I is crucial for viral DNA replication. G4 pull-down analysis identifies viral DNA replication factors, such as IE2, UL84, and UL44, as G4-binding proteins. In enzyme-linked immunosorbent assays, specific G4-binding ligands inhibit G4 binding by the viral proteins. The Epstein-Barr virus oriLyt core element also forms a stable G4 that could substitute for the oriLyt ER-I G4 in HCMV. These results demonstrate that viral G4s in replication origins represent an essential structural element in recruiting replication factors and might be a therapeutic target against viral infections., (© 2024. The Author(s).)

Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti . Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriV s of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC , and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 blaNDM-1 gene and its variants encode metallo-beta-lactamases that confer resistance to almost all beta-lactam antibiotics. Genes encoding blaNDM-1 and its variants can be found in several Acinetobacter species, and they are usually linked to two different plasmid clades. The plasmids in one of these clades contain a gene encoding a Rep protein of the Rep_3 superfamily. The other clade consists of medium-sized plasmids in which the gene (s) involved in plasmid replication initiation (rep)have not yet been identified. In the present study, we identified the minimal replication region of a blaNDM-1-carrying plasmid of Acinetobacter haemolyticus AN54 (pAhaeAN54e), a member of this second clade. This region of 834 paired bases encodes three small peptides, all of which have roles in plasmid maintenance. The plasmids containing this minimal replication region are closely related; almost all contain blaNDM genes, and they are found in multiple Acinetobacter species, including A. baumannii. None of these plasmids contain an annotated Rep gene, suggesting that their replication relies on the minimal replication region that they share with the plasmid pAhaeAN54e. These observations suggest that this plasmid lineage plays a crucial role in the dissemination of the blaNDM-1 gene and its variants., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bello-López 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.)

Horizontal gene transfer (HGT) is a powerful evolutionary force that considerably shapes the structure of prokaryotic genomes and is associated with genomic islands (GIs). A GI is a DNA segment composed of transferred genes that can be found within a prokaryotic genome, obtained through HGT. Much research has focused on detecting GIs in genomes, but here we pursue a new course, which is identifying possible preferred locations of GIs in the prokaryotic genome. Here, we identify the locations of the GIs within prokaryotic genomes to examine patterns in those locations. Prokaryotic GIs were analyzed according to the genome structure that they are located in, whether it be a circular or a linear genome. The analytical investigations employed are: (1) studying the GI locations in relation to the origin of replication (oriC); (2) exploring the distances between GIs; and (3) determining the distribution of GIs across the genomes. For each of the investigations, the analysis was performed on all of the GIs in the data set. Moreover, to void bias caused by the distribution of the genomes represented, the GIs in one genome from each species and the GIs of the most frequent species are also analyzed. Overall, the results showed that there are preferred sites for the GIs in the genome. In the linear genomes, these sites are usually located in the oriC region and terminus region, while in the circular genomes, they are located solely in the terminus region. These results also showed that the distance distribution between the GIs is almost exponential, which proves that GIs have preferred sites within genomes. The oriC and termniuns are preferred sites for the GIs and a possible natural explanation for this could be connected to the content of the oriC region. Moreover, the content of the GIs in terms of its protein families was studied and the results demonstrated that the majority of frequent protein families are close to identical in each section., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Aldaihani, Heath. 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.)

Plasmids, despite their critical role in antibiotic resistance and modern biotechnology, are understood in only a few bacterial groups in terms of their natural ecological dynamics. The bacterial phylum Planctomycetes, known for its unique molecular and cellular biology, has a largely unexplored plasmidome. This study offers a thorough exploration of the diversity of natural plasmids within Planctomycetes, which could serve as a foundation for developing various genetic research tools for this phylum. Planctomycetes plasmids encode a broad range of biological functions and appear to have coevolved significantly with their host chromosomes, sharing many homologues. Recent transfer events of insertion sequences between cohabiting chromosomes and plasmids were also observed. Interestingly, 64% of plasmid genes are distantly related to either chromosomally encoded genes or have homologues in plasmids from other bacterial groups. The planctomycetal plasmidome is composed of 36% exclusive proteins. Most planctomycetal plasmids encode a replication initiation protein from the Replication Protein A family near a putative iteron-containing replication origin, as well as active type I partition systems. The identification of one conjugative and three mobilizable plasmids suggests the occurrence of horizontal gene transfer via conjugation within this phylum. This comprehensive description enhances our understanding of the plasmidome of Planctomycetes and its potential implications in antibiotic resistance and biotechnology., (© 2024 The Authors. Environmental Microbiology published by John Wiley & Sons Ltd.)

Gene-strand bias is a characteristic feature of bacterial genome organization wherein genes are preferentially encoded on the leading strand of replication, promoting co-orientation of replication and transcription. This co-orientation bias has evolved to protect gene essentiality, expression, and genomic stability from the harmful effects of head-on replication-transcription collisions. However, the origin, variation, and maintenance of gene-strand bias remain elusive. Here, we reveal that the frequency of inversions that alter gene orientation exhibits large variation across bacterial populations and negatively correlates with gene-strand bias. The density, distance, and distribution of inverted repeats show a similar negative relationship with gene-strand bias explaining the heterogeneity in inversions. Importantly, these observations are broadly evident across the entire bacterial kingdom uncovering inversions and inverted repeats as primary factors underlying the variation in gene-strand bias and its maintenance. The distinct catalytic subunits of replicative DNA polymerase have co-evolved with gene-strand bias, suggesting a close link between replication and the origin of gene-strand bias. Congruently, inversion frequencies and inverted repeats vary among bacteria with different DNA polymerases. In summary, we propose that the nature of replication determines the fitness cost of replication-transcription collisions, establishing a selection gradient on gene-strand bias by fine-tuning DNA sequence repeats and, thereby, gene inversions., (© 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.)

Various origin mapping approaches have enabled genome-wide identification of origins of replication (ORI) in model organisms, but only a few studies have focused on divergent organisms. By employing three complementary approaches we provide a high-resolution map of ORIs in Plasmodium falciparum, the deadliest human malaria parasite. We profiled the distribution of origin of recognition complex (ORC) binding sites by ChIP-seq of two PfORC subunits and mapped active ORIs using NFS and SNS-seq. We show that ORIs lack sequence specificity but are not randomly distributed, and group in clusters. Licensing is biased towards regions of higher GC content and associated with G-quadruplex forming sequences (G4FS). While strong transcription likely enhances firing, active origins are depleted from transcription start sites. Instead, most accumulate in transcriptionally active gene bodies. Single molecule analysis of nanopore reads containing multiple initiation events, which could have only come from individual nuclei, showed a relationship between the replication fork pace and the distance to the nearest origin. While some similarities were drawn with the canonic eukaryote model, the distribution of ORIs in P. falciparum is likely shaped by unique genomic features such as extreme AT-richness-a product of evolutionary pressure imposed by the parasitic lifestyle., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Biological cells replicate their genomes in a well-planned manner. The DNA replication program of an organism determines the timing at which different genomic regions are replicated, with fundamental consequences for cell homeostasis and genome stability. In a growing cell culture, genomic regions that are replicated early should be more abundant than regions that are replicated late. This abundance pattern can be experimentally measured using deep sequencing. However, a general quantitative theory linking this pattern to the replication program is still lacking. In this paper, we predict the abundance of DNA fragments in asynchronously growing cultures from any given stochastic model of the DNA replication program. As key examples, we present stochastic models of the DNA replication programs in budding yeast and Escherichia coli. In both cases, our model results are in excellent agreement with experimental data and permit to infer key information about the replication program. In particular, our method is able to infer the locations of known replication origins in budding yeast with high accuracy. These examples demonstrate that our method can provide insight into a broad range of organisms, from bacteria to eukaryotes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pflug 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 mammalian DNA replication timing (RT) program is crucial for the proper functioning and integrity of the genome. The best-known mechanism for controlling RT is the suppression of late origins of replication in heterochromatin by RIF1. Here, we report that in antigen-activated, hypermutating murine B lymphocytes, RIF1 binds predominantly to early-replicating active chromatin and promotes early replication, but plays a minor role in regulating replication origin activity, gene expression and genome organization in B cells. Furthermore, we find that RIF1 functions in a complementary and non-epistatic manner with minichromosome maintenance (MCM) proteins to establish early RT signatures genome-wide and, specifically, to ensure the early replication of highly transcribed genes. These findings reveal additional layers of regulation within the B cell RT program, driven by the coordinated activity of RIF1 and MCM proteins., (© 2023. The Author(s).)

In eukaryotic genomes, hundreds to thousands of potential start sites of DNA replication named origins are dispersed across each of the linear chromosomes. During S-phase, only a subset of origins is selected in a stochastic manner to assemble bidirectional replication forks and initiate DNA synthesis. Despite substantial progress in our understanding of this complex process, a comprehensive 'identity code' that defines origins based on specific nucleotide sequences, DNA structural features, the local chromatin environment, or 3D genome architecture is still missing. In this article, we review the genetic and epigenetic features of replication origins in yeast and metazoan chromosomes and highlight recent insights into how this flexibility in origin usage contributes to nuclear organization, cell growth, differentiation, and genome stability.

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium., (© 2023. The Author(s).)

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Our data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

FAM111A is a replisome-associated protein and dominant mutations within its trypsin-like peptidase domain are linked to severe human developmental syndrome, the Kenny-Caffey syndrome. However, FAM111A functions remain unclear. Here, we show that FAM111A facilitates efficient activation of DNA replication origins. Upon hydroxyurea treatment, FAM111A-depleted cells exhibit reduced single-stranded DNA formation and a better survival rate. Unrestrained expression of FAM111A WT and patient mutants causes accumulation of DNA damage and cell death, only when the peptidase domain remains intact. Unrestrained expression of FAM111A WT also causes increased single-stranded DNA formation that relies on S phase entry, FAM111A peptidase activity but not its binding to proliferating cell nuclear antigen. Altogether, these data unveil how FAM111A promotes DNA replication under normal conditions and becomes harmful in a disease context., (© 2023 Rios-Szwed et al.)

Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a 'super regulatory center' in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries., (© 2023. Springer Nature Limited.)

Replication of vertebrate genomes is tightly regulated to ensure accurate duplication, but our understanding of the interplay between genetic and epigenetic factors in this regulation remains incomplete. Here, we investigated the involvement of three elements enriched at gene promoters and replication origins: guanine-rich motifs potentially forming G-quadruplexes (pG4s), nucleosome-free regions (NFRs), and the histone variant H2A.Z, in the firing of origins of replication in vertebrates. We show that two pG4s on the same DNA strand (dimeric pG4s) are sufficient to induce the assembly of an efficient minimal replication origin without inducing transcription in avian DT40 cells. Dimeric pG4s in replication origins are associated with formation of an NFR next to precisely-positioned nucleosomes enriched in H2A.Z on this minimal origin and genome-wide. Thus, our data suggest that dimeric pG4s are important for the organization and duplication of vertebrate genomes. It supports the hypothesis that a nucleosome close to an NFR is a shared signal for the formation of replication origins in eukaryotes., (© 2023. Springer Nature Limited.)

Origins of replication are genomic regions in which replication initiates in a bidirectional manner. Recently, a new methodology (origin-derived single-stranded DNA sequencing; ori-SSDS) was developed that allows the detection of replication initiation in a strand-specific manner. Reanalysis of the strand-specific data revealed that 18-33% of the peaks are non-symmetrical, suggesting a single direction of replication. Analysis of replication fork direction data revealed that these are origins of replication in which the replication is paused in one of the directions, probably due to the existence of a replication fork barrier. Analysis of the unidirectional origins revealed a preference of G4 quadruplexes for the blocked leading strand. Taken together, our analysis identified hundreds of genomic locations in which the replication initiates only in one direction, and suggests that G4 quadruplexes may serve as replication fork barriers in such places.

In human and other metazoans, the determinants of replication origin location and strength are still elusive. Origins are licensed in G1 phase and fired in S phase of the cell cycle, respectively. It is debated which of these two temporally separate steps determines origin efficiency. Experiments can independently profile mean replication timing (MRT) and replication fork directionality (RFD) genome-wide. Such profiles contain information on multiple origins' properties and on fork speed. Due to possible origin inactivation by passive replication, however, observed and intrinsic origin efficiencies can markedly differ. Thus, there is a need for methods to infer intrinsic from observed origin efficiency, which is context-dependent. Here, we show that MRT and RFD data are highly consistent with each other but contain information at different spatial scales. Using neural networks, we infer an origin licensing landscape that, when inserted in an appropriate simulation framework, jointly predicts MRT and RFD data with unprecedented precision and underlies the importance of dispersive origin firing. We furthermore uncover an analytical formula that predicts intrinsic from observed origin efficiency combined with MRT data. Comparison of inferred intrinsic origin efficiencies with experimental profiles of licensed origins (ORC, MCM) and actual initiation events (Bubble-seq, SNS-seq, OK-seq, ORM) show that intrinsic origin efficiency is not solely determined by licensing efficiency. Thus, human replication origin efficiency is set at both the origin licensing and firing steps., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Arbona 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 malaria parasite Plasmodium falciparum replicates via schizogony: an unusual type of cell cycle involving asynchronous replication of multiple nuclei within the same cytoplasm. Here, we present the first comprehensive study of DNA replication origin specification and activation during Plasmodium schizogony. Potential replication origins were abundant, with ORC1-binding sites detected every ∼800 bp. In this extremely A/T-biased genome, the sites were biased towards areas of higher G/C content, and contained no specific sequence motif. Origin activation was then measured at single-molecule resolution using newly developed DNAscent technology: a powerful method of detecting replication fork movement via base analogues in DNA sequenced on the Oxford Nanopore platform. Unusually, origins were preferentially activated in areas of low transcriptional activity, and replication forks also moved fastest through lowly transcribed genes. This contrasts with the way that origin activation is organised in other systems, such as human cells, and suggests that P. falciparum has evolved its S-phase specifically to minimise conflicts between transcription and origin firing. This may be particularly important to maximise the efficiency and accuracy of schizogony, with its multiple rounds of DNA replication and its absence of canonical cell-cycle checkpoints., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Enterococcal plasmids have attracted considerable interest because of their indispensable role in the pathogenesis and dissemination of multidrug-resistance. In this work, five novel plasmids pSRB2, pSRB3, pSRB4, pSRB5 and pSRB7 have been identified and characterised, coexisting in Eneterococcus italicus SD1 from fermented milk. The plasmids pSRB2, pSRB3 and pSRB5 were found to replicate via theta mode of replication while pSRB4 and pSRB7 were rolling-circle plasmids. Comparative analysis of SD1-plasmids dictated that the plasmids are mosaic with novel architecture. Plasmids pSRB2 and pSRB5 are comprised of a typical iteron-based class-A theta type origin of replication, whereas pSRB3 has a Class-D theta type replication origin like pAMβ1. The plasmids pSRB4 and pSRB7 shared similar ori as in pWV01. The SD1 class-A theta type plasmids shared significant homology between their replication proteins with differences in their DNA-binding domain and comprises of distinct iterons. The differences in their iterons and replication proteins restricts the "handcuff" formation for inhibition of plasmid replication, rendering to their compatibility to coexist. Similarly, for SD1 rolling circle plasmids the differences in the replication protein binding site in the origin and the replication protein supports their coexistence by inhibiting the crosstalk between the origins and replication proteins. The phylogenetic tree of their replication proteins revealed their distant kinship. The results indicate that the identified plasmids are unique to E. italicus SD1, providing further opportunities to study their utility in designing multiple gene expression systems for the simultaneous production of proteins in enterococci with the renewed concept of plasmid incompatibility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

DoriC was first launched in 2007 as a database of replication origins (oriCs) in bacterial genomes and has since been constantly updated to integrate the latest research progress in this field. The database was subsequently extended to include the oriCs in archaeal genomes as well as those in plasmids. This latest release, DoriC 12.0, includes the oriCs in both draft and complete prokaryotic genomes. At the same time, the number of oriCs in the database has also increased significantly and currently contains over 200 000 bacterial entries distributed in more than 40 phyla. Among them, a large number are from bacteria in new phyla whose oriCs were not explored before. Additionally, new oriC features and improvements have been introduced, especially in the visualization and analysis of oriCs. Currently, DoriC is considered as an important database in the fields of bioinformatics, microbial genomics, and even synthetic biology, providing a valuable resource as well as a comprehensive platform for the research on oriCs. DoriC 12.0 can be accessed at https://tubic.org/doric/ and http://tubic.tju.edu.cn/doric/., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chromosomal instability (CIN) is a hallmark of cancer and comprises structural CIN (S-CIN) and numerical or whole chromosomal CIN (W-CIN). Recent work indicated that replication stress (RS), known to contribute to S-CIN, also affects mitotic chromosome segregation, possibly explaining the common co-existence of S-CIN and W-CIN in human cancer. Here, we show that RS-induced increased origin firing is sufficient to trigger W-CIN in human cancer cells. We discovered that overexpression of origin firing genes, including GINS1 and CDC45, correlates with W-CIN in human cancer specimens and causes W-CIN in otherwise chromosomally stable human cells. Furthermore, modulation of the ATR-CDK1-RIF1 axis increases the number of firing origins and leads to W-CIN. Importantly, chromosome missegregation upon additional origin firing is mediated by increased mitotic microtubule growth rates, a mitotic defect prevalent in chromosomally unstable cancer cells. Thus, our study identifies increased replication origin firing as a cancer-relevant trigger for chromosomal instability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The structural arrangements of bacterial chromosomes vary widely between closely related species and can result in significant phenotypic outcomes. The appearance of large-scale chromosomal inversions that are symmetric relative to markers for the origin of replication ( OriC ) has been previously observed; however, the overall prevalence of replication-associated structural rearrangements (RASRs) in bacteria and their causal mechanisms are currently unknown. Here, we systematically identify the locations of RASRs in species with multiple complete-sequenced genomes and investigate potential mediating biological mechanisms. We found that 247 of 313 species contained sequences with at least one large (>50 Kb) inversion in their sequence comparisons, and the aggregated inversion distances away from symmetry were normally distributed with a mean of zero. Many inversions that were offset from dnaA were found to be centered on a different marker for the OriC Instances of flanking repeats provide evidence that breaks formed during the replication process could be repaired to opposing positions. We also found a strong relationship between the later stages of replication and the range in distance variation from symmetry., (© 2022 D’Iorio and Dewar.)

Eukaryotic chromosomes are organized into structural and functional domains with characteristic replication timings, which are thought to contribute to epigenetic programming and genome stability. Differential replication timing results from epigenetic mechanisms that positively and negatively regulate the competition for limiting replication initiation factors. Histone deacetylase Sir2 negatively regulates initiation of the multicopy (∼150) rDNA origins, while Rpd3 histone deacetylase negatively regulates firing of single-copy origins. However, Rpd3's effect on single-copy origins might derive indirectly from a positive function for Rpd3 in rDNA origin firing shifting the competitive balance. Our quantitative experiments support the idea that origins compete for limiting factors; however, our results show that Rpd3's effect on single-copy origin is independent of rDNA copy-number and of Sir2's effects on rDNA origin firing. Whereas RPD3 deletion and SIR2 deletion alter the early S phase dynamics of single-copy and rDNA origin firings in opposite fashion, unexpectedly only RPD3 deletion suppresses overall rDNA origin efficiency across S phase. Increased origin activation in rpd3Δ requires Fkh1/2, suggesting that Rpd3 opposes Fkh1/2-origin stimulation, which involves recruitment of Dbf4-dependent kinase (DDK). Indeed, Fkh1 binding increases at Rpd3-regulated origins in rpd3Δ cells in G1, supporting a mechanism whereby Rpd3 influences initiation timing of single-copy origins directly through modulation of Fkh1-origin binding. Genetic suppression of a DBF4 hypomorphic mutation by RPD3 deletion further supports the conclusion that Rpd3 impedes DDK recruitment by Fkh1, revealing a mechanism of Rpd3 in origin regulation.

Replication and segregation of the genetic information is necessary for a cell to proliferate. In Bacillus subtilis , the Par system (ParA/Soj, ParB/Spo0J and parS ) is required for segregation of the chromosome origin ( oriC ) region and for proper control of DNA replication initiation. ParB binds parS sites clustered near the origin of replication and assembles into sliding clamps that interact with ParA to drive origin segregation through a diffusion-ratchet mechanism. As part of this dynamic process, ParB stimulates ParA ATPase activity to trigger its switch from an ATP-bound dimer to an ADP-bound monomer. In addition to its conserved role in DNA segregation, ParA is also a regulator of the master DNA replication initiation protein DnaA. We hypothesized that in B. subtilis the location of the Par system proximal to oriC would be necessary for ParA to properly regulate DnaA. To test this model, we constructed a range of genetically modified strains with altered numbers and locations of parS sites, many of which perturbed chromosome origin segregation as expected. Contrary to our hypothesis, the results show that regulation of DNA replication initiation by ParA is maintained when a parS site is separated from oriC . Because a single parS site is sufficient for proper control of ParA, the results are consistent with a model where ParA is efficiently regulated by ParB sliding clamps following loading at parS .

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).)

Efficient production of biochemicals and proteins in cell factories frequently benefits from a two-stage bioprocess in which growth and production phases are decoupled. Here, we describe a novel growth switch based on the permanent removal of the origin of replication ( oriC ) from the Escherichia coli chromosome. Without oriC , cells cannot initiate a new round of replication, and they stop growing while their metabolism remains active. Our system relies on a serine recombinase from bacteriophage phiC31 whose expression is controlled by the temperature-sensitive cI857 repressor from phage lambda. The reporter protein expression in switched cells continues after cessation of growth, leading to protein levels up to 5 times higher compared to nonswitching cells. Switching induces a unique physiological state that is different from both normal exponential and stationary phases. The switched cells remain in this state even when not growing, retain their protein synthesis capacity, and do not induce proteins associated with the stationary phase. Our switcher technology is potentially useful for a range of products and applicable in many bacterial species for decoupling growth and production.

Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered 'heavy-light' (HL) by incorporation of BrdUTP while unreplicated DNA remains 'light-light' (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic DNA replication is regulated by conserved mechanisms that bring about a spatial and temporal organization in which distinct genomic domains are copied at characteristic times during S phase. Although this replication program has been closely linked with genome architecture, we still do not understand key aspects of how chromosomal context modulates the activity of replication origins. To address this question, we have exploited models that combine engineered genomic rearrangements with the unique replication programs of post-quiescence and pre-meiotic S phases. Our results demonstrate that large-scale inversions surprisingly do not affect cell proliferation and meiotic progression, despite inducing a restructuring of replication domains on each rearranged chromosome. Remarkably, these alterations in the organization of DNA replication are entirely due to changes in the positions of existing origins along the chromosome, as their efficiencies remain virtually unaffected genome wide. However, we identified striking alterations in origin firing proximal to the fusion points of each inversion, suggesting that the immediate chromosomal neighborhood of an origin is a crucial determinant of its activity. Interestingly, the impact of genome reorganization on replication initiation is highly comparable in the post-quiescent and pre-meiotic S phases, despite the differences in DNA metabolism in these two physiological states. Our findings therefore shed new light on how origin selection and the replication program are governed by chromosomal architecture.

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

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability 1,2 . At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs) 3-6 , subTADs 7 and loops 8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase., (© 2022. The Author(s).)

Partition systems are widespread among bacterial chromosomes. They are composed of two effectors, ParA and ParB, and cis acting sites, parS , located close to the replication origin of the chromosome ( oriC ). ParABS participate in chromosome segregation, at least in part because they serve to properly position sister copies of oriC . A fourth element, located at cell poles, is also involved in some cases, such as HubP for the ParABS1 system of Vibrio cholerae chromosome 1 (ch1). The polar anchoring of oriC of ch1 ( oriC1 ) is lost when HubP or ParABS1 are inactivated. Here, we report that in the absence of HubP, ParABS1 actively maintains oriC1 at mid-cell, leading to the subcellular separation of the two ch1 replication arms. We further show that parS1 sites ectopically inserted in chromosome 2 (ch2) stabilize the inheritance of this replicon in the absence of its endogenous partition system, even without HubP. We also observe the positioning interference between oriC1 and oriC of ch2 regions when their positionings are both driven by ParABS1. Altogether, these data indicate that ParABS1 remains functional in the absence of HubP, which raises questions about the role of the polar anchoring of oriC1 in the cell cycle.

Protein function often requires remodeling of protein structure. In the well-studied iteron-containing plasmids, the initiator of replication has a dimerization interface that undergoes chaperone-mediated remodeling. This remodeling reduces dimerization and promotes DNA replication, since only monomers bind origin DNA. A structurally homologs interface exists in RctB, the replication initiator of Vibrio cholerae chromosome 2 (Chr2). Chaperones also promote Chr2 replication, although both monomers and dimers of RctB bind to origin, and chaperones increase the binding of both. Here we report how five changes in the dimerization interface of RctB affect the protein. The mutants are variously defective in dimerization, more active as initiator, and except in one case, unresponsive to chaperone (DnaJ). The results indicate that chaperones also reduce RctB dimerization and support the proposal that the paradoxical chaperone-promoted dimer binding likely represents sequential binding of monomers on DNA. RctB is also activated for replication initiation upon binding to a DNA site, crtS, and three of the mutants are also unresponsive to crtS. This suggests that crtS, like chaperones, reduces dimerization, but additional evidence suggests that the remodelling activities function independently. Involvement of two remodelers in reducing dimerization signifies the importance of dimerization in limiting Chr2 replication., (Published by Oxford University Press on behalf of Nucleic Acids Research 2022.)

DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We develop an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes in silico flow cytometry, a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyze up to 2437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression., (© 2022. The Author(s).)

The chromatin-based rule governing the selection and activation of replication origins remains to be elucidated. It is believed that DNA replication initiates from open chromatin domains; thus, replication origins reside in open and active chromatin. However, we report here that lysine-specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation favoring chromatin condensation, interacts with the DNA replication machinery in human cells. We find that LSD1 level peaks in early S phase, when it is required for DNA replication by facilitating origin firing in euchromatic regions. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for the pre-replicative complex (pre-RC) binding. Remarkably, LSD1 deficiency leads to a genome-wide switch of replication from early to late. We show that LSD1-engaged DNA replication is mechanistically linked to the loading of TopBP1-Interacting Checkpoint and Replication Regulator (TICRR) onto the pre-RC and subsequent recruitment of CDC45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing, highlighting the importance of epigenetic regulation in the activation of replication origins. As selective inhibitors of LSD1 are being exploited as potential cancer therapeutics, our study supports the importance of leveraging an appropriate level of LSD1 to curb the side effects of anti-LSD1 therapy., (© 2022. The Author(s).)

Dbf4 is the cyclin-like subunit for the Dbf4-dependent protein kinase (DDK), required for activating the replicative helicase at DNA replication origin that fire during S phase. Dbf4 also functions as an adaptor, targeting the DDK to different groups of origins and substrates. Here we report a genome-wide analysis of origin firing in a budding yeast mutant, dbf4-zn, lacking the Zn 2+ finger domain within the C-terminus of Dbf4. At one group of origins, which we call dromedaries, we observe an unanticipated DNA replication phenotype: accumulation of single-stranded DNA spanning ± 5kbp from the center of the origins. A similar accumulation of single-stranded DNA at origins occurs more globally in pri1-m4 mutants defective for the catalytic subunit of DNA primase and rad53 mutants defective for the S phase checkpoint following DNA replication stress. We propose the Dbf4 Zn 2+ finger suppresses single-stranded gaps at replication forks emanating from dromedary origins. Certain origins may impose an elevated requirement for the DDK to fully initiate DNA synthesis following origin activation. Alternatively, dbf4-zn may be defective for stabilizing/restarting replication forks emanating from dromedary origins during replication stress., (© 2022. The Author(s).)

Streptomyces linear plasmids often contain an internal replication origin. In this study, a new replication origin was identified and confirmed in the 1.8-Mb plasmid pSCATT of Streptomyces cattleya DSM46488. The real-time qPCR results indicated that the copy number of pSCATT was one copy per chromosome. The identified replication origin oriC1-II was found to locate in the central region of pSCATT and was 2 kb in size. This replication origin consists of a protein-coding gene SCATT_p08010 with an unknown function and the upstream non-coding sequence. Deletion or disruption analysis of SCATT_p08010 or the upstream non-coding sequence revealed that both SCATT_p08010 and the non-coding sequence were essential for replication. However, the identified replication origin was shown to endow the plasmid with the ability to replicate in a circular model but not in a linear model in S. lividans. Interestingly, the knockout of the replication origin did not result in the curing of pSCATT, indicating that there might be other replication origins present in the mega-plasmid. The experimental validation of the central replication origin oriC1-II might be helpful for the investigation of the replication mechanism of the mega-plasmid and the genome evolution of Streptomyces., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Fundamental to our understanding of chromosome duplication is the idea that replication origins function both as sites where MCM helicases are loaded during the G1 phase and where synthesis begins in S phase. However, the temporal delay between phases exposes the replisome assembly pathway to potential disruption prior to replication. Using multicolor, single-molecule imaging, we systematically study the consequences of encounters between actively transcribing RNA polymerases (RNAPs) and replication initiation intermediates in the context of chromatin. We demonstrate that RNAP can push multiple licensed MCM helicases over long distances with nucleosomes ejected or displaced. Unexpectedly, we observe that MCM helicase loading intermediates also can be repositioned by RNAP and continue origin licensing after encounters with RNAP, providing a web of alternative origin specification pathways. Taken together, our observations reveal a surprising mobility in origin-licensing factors that confers resistance to the complex challenges posed by diverse obstacles encountered on chromosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype., Competing Interests: The authors have declared that no competing interests exist.