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DNA polymerase Δ synthesizes both strands during break-induced replication

ABSTRACT: We applied our ribonucleotide mapping technique HydEn-seq to study DNA polymerase usage during break-induced replication (BIR) in the budding yeast Saccharomyces cerevisiae.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE133558 | GEO | 2019/07/01

REPOSITORIES: GEO

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DNA Polymerase Delta Synthesizes Both Strands during Break-Induced Replication.

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Evidence that DNA polymerase δ contributes to initiation of leading strand DNA replication in Saccharomyces cerevisiae

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Project description:Antimetabolite chemotherapies increase uracil levels in DNA, and thus identification of factors that influence the uracil content in DNA may have implications for understanding uracil-mediated chromosomal instability. We previously showed in the budding yeast Saccharomyces cerevisiae that uracil content in DNA correlates with replication timing, where the earliest and latest replicating regions are depleted in uracil. Here, we manipulated nucleotide biosynthesis enzymes in budding yeast to determine whether the pattern of uracil incorporation could be altered. In strains with high levels of uracil incorporation, deletion of dCMP deaminase (Dcd1) accelerated uracil incorporation at early-firing origins, likely due to rapid dTTP pool depletion. In contrast, increasing the activity of ribonucleotide reductase, which is required for the synthesis of all dNTPs via ribonucleotide diphosphates, lead to dUTP and dTTP pool equilibration and a concomitant increase in uracil content throughout the genome. These data suggest that uracil availability and the dUTP:dTTP ratio are temporally regulated during S phase and govern uracil incorporation into the genome. Therapeutic manipulation of nucleotide biosynthesis in human cells to either increase the dUTP pool or deplete the dTTP pool in early S phase may therefore improve the efficacy of antimetabolite chemotherapies.

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plasmid-based BIR screen in S. cerevisiae

Project description:We used a plamsid-based assay to identify novel S. cerevisiae mutants with abnormal Break Induced Replication (BIR) efficiencies. We pooled the ca. 5000 yeast deletion mutants from the systematic deletion library and compared the relative tranformation efficiencies of a circular versus linear truncated minichromosome using tag arrays. Our main result is the identification of Fun30 as a novel chromatin remodelr facilitating DNA end resection.

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