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

ABSTRACT: 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.

SUBMITTER: Gispan A

PROVIDER: S-EPMC5287236 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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

Gispan Ariel A Carmi Miri M Barkai Naama N

Genome research 20161227 2

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 b ...[more]

PMID: 28028072

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Project description:Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.

Project description:Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096-22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825-831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.

Dataset Information

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

Publications

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

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