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DNA molecular combing-based replication fork directionality profiling.

ABSTRACT: The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

SUBMITTER: Blin M

PROVIDER: S-EPMC8266662 | biostudies-literature |

REPOSITORIES: biostudies-literature

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DNA molecular combing-based replication fork directionality profiling

Project description:The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas ineffective origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing (OK-seq). Importantly, we demonstrate that inefficient origins do not manifest as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

2021-03-16 | GSE164252 | GEO

DNA molecular combing-based replication fork directionality profiling

Project description:DNA molecular combing-based replication fork directionality profiling

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OKseqHMM: a genome-wide replication fork directionality analysis toolkit

Project description:During each cell division, tens of thousands of DNA replication origins are coordinately activated to ensure the complete duplication of the entire human genome. However, the progression of replication forks can be challenged by numerous factors. One such factor is transcription-replication conflict (TRC), which can either be co-directional or head-on and the latter has been revealed as more dangerous for genome integrity. In order to study the direction of replication fork movement and TRC, we developed a bioinformatics tool, called OKseqHMM, to direct measure the replication fork directionality (RFD) as well as replication initiation and termination, along human genome obtained by sequencing of Okazaki fragments (OK-Seq) and related techniques. We have gathered and analyzed OK-seq data from a large number of organisms including yeast, mouse and human, to generate high-quality RFD profiles and determine initiation zones and termination zones by using Hidden Markov Model (HMM) algorithm (all tools and data are available at https://github.com/CL-CHEN-Lab/OK-Seq). In addition, we have extended our analysis to data obtained by related techniques, such as eSPAN and TrAEL-seq, which also contain RFD information. Our works, therefore, provide an important tool and resource for the community to further study TRC and genome instability, in a wide range of cell line models and growth conditions, which is of prime importance for human health.

2022-12-31 | GSE193547 | GEO

Model-based analysis of DNA replication profiles: predicting replication fork velocity and initiation rate by profiling free-cycling cells.

Project description:Eukaryotic cells initiate DNA synthesis by sequential firing of hundreds of origins. This ordered replication is described by replication profiles, which measure the DNA content within a cell population. Here, we show that replication dynamics can be deduced from replication profiles of free-cycling cells. While such profiles lack explicit temporal information, they are sensitive to fork velocity and initiation capacity through the passive replication pattern, namely the replication of origins by forks emanating elsewhere. We apply our model-based approach to a compendium of profiles that include most viable budding yeast mutants implicated in replication. Predicted changes in fork velocity or initiation capacity are verified by profiling synchronously replicating cells. Notably, most mutants implicated in late (or early) origin effects are explained by global modulation of fork velocity or initiation capacity. Our approach provides a rigorous framework for analyzing DNA replication profiles of free-cycling cells.

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Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

Project description:The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

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RMI1 promotes DNA replication fork progression and recovery from replication fork stress.

Project description:RMI1 is a member of an evolutionarily conserved complex composed of BLM and topoisomerase III? (TopoIII?). This complex exhibits strand passage activity in vitro, which is likely important for DNA repair and DNA replication in vivo. The inactivation of RMI1 causes genome instability, including elevated levels of sister chromatid exchange and accelerated tumorigenesis. Using molecular combing to analyze DNA replication at the single-molecule level, we show that RMI1 is required to promote normal replication fork progression. The fork progression defect in RMI1-depleted cells is alleviated in cells lacking BLM, indicating that RMI1 functions downstream of BLM in promoting replication elongation. RMI1 localizes to subnuclear foci with BLM and TopoIII? in response to replication stress. The proper localization of the complex requires a BLM-TopoIII?-RMI1 interaction and is essential for RMI1 to promote recovery from replication stress. These findings reveal direct roles of RMI1 in DNA replication and the replication stress response, which could explain the molecular basis for its involvement in suppressing sister chromatid exchange and tumorigenesis.

| S-EPMC3434523 | biostudies-literature

DNA replication stress triggers rapid DNA replication fork breakage by Artemis and XPF.

Project description:DNA replication stress (DRS) leads to the accumulation of stalled DNA replication forks leaving a fraction of genomic loci incompletely replicated, a source of chromosomal rearrangements during their partition in mitosis. MUS81 is known to limit the occurrence of chromosomal instability by processing these unresolved loci during mitosis. Here, we unveil that the endonucleases ARTEMIS and XPF-ERCC1 can also induce stalled DNA replication forks cleavage through non-epistatic pathways all along S and G2 phases of the cell cycle. We also showed that both nucleases are recruited to chromatin to promote replication fork restart. Finally, we found that rapid chromosomal breakage controlled by ARTEMIS and XPF is important to prevent mitotic segregation defects. Collectively, these results reveal that Rapid Replication Fork Breakage (RRFB) mediated by ARTEMIS and XPF in response to DRS contributes to DNA replication efficiency and limit chromosomal instability.

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Polymerase dynamics at the eukaryotic DNA replication fork.

Project description:This review discusses recent insights in the roles of DNA polymerases (Pol) delta and epsilon in eukaryotic DNA replication. A growing body of evidence specifies Pol epsilon as the leading strand DNA polymerase and Pol delta as the lagging strand polymerase during undisturbed DNA replication. New evidence supporting this model comes from the use of polymerase mutants that show an asymmetric mutator phenotype for certain mispairs, allowing an unambiguous strand assignment for these enzymes. On the lagging strand, Pol delta corrects errors made by Pol alpha during Okazaki fragment initiation. During Okazaki fragment maturation, the extent of strand displacement synthesis by Pol delta determines whether maturation proceeds by the short or long flap processing pathway. In the more common short flap pathway, Pol delta coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase.

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DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI.

Project description:It is generally believed that DNA replication in most eukaryotes proceeds according to a precise program in which there is a defined temporal order by which each chromosomal region is duplicated. However, the regularity of this program at the level of individual chromosomes, in terms of both the relative timing and the size of the DNA domain, has not been addressed. Here, the replication of chromosome VI from synchronized budding yeast was studied at a resolution of approximately 1 kb with DNA combing and fluorescence microscopy. Contrary to what would be expected from cells following a rigorous temporal program, no two molecules exhibited the same replication pattern. Moreover, a direct evaluation of the extent to which the replication of distant chromosomal segments was coordinated indicates that the overwhelming majority of these segments were replicated independently. Importantly, averaging the patterns of all the fibers examined recapitulates the ensemble-averaged patterns obtained from population studies of the replication of chromosome VI. Thus, rather than an absolutely defined temporal order of replication, replication timing appears to be essentially probabilistic within individual cells, exhibiting only temporal tendencies within extended domains.

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The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest.

Project description:Werner syndrome is an autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several lines of evidence have suggested that the Werner syndrome protein WRN plays a role in DNA replication and S-phase progression. In order to define the exact role of WRN in genomic replication we examined cell cycle kinetics during normal cell division and after methyl-methane-sulfonate (MMS) DNA damage or hydroxyurea (HU)-mediated replication arrest following acute depletion of WRN from human fibroblasts. Loss of WRN markedly extended the time cells needed to complete the cell cycle after either of these genotoxic treatments. Moreover, replication track analysis of individual, stretched DNA fibers showed that WRN depletion significantly reduced the speed at which replication forks elongated in vivo after MMS or HU treatment. These results establish the importance of WRN during genomic replication and indicate that WRN acts to facilitate fork progression after DNA damage or replication arrest. The data provide a mechanistic basis for a better understanding of WRN-mediated maintenance of genomic stability and for predicting the outcomes of DNA-targeting chemotherapy in several adult cancers that silence WRN expression.

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