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Mapping polymerase usage by tracking ribonucleotide incorporation (HydEn-seq) during DNA replication

ABSTRACT: We devised and improved on our hydrolytic end sequencing (HydEn-seq) that mapping ribonucleotide incorporation in genome and used this method to track DNA replicative polymerase usage. We uncovered striking exceptions to canonical polymerase division of labor in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

ORGANISM(S): Schizosaccharomyces pombe Saccharomyces cerevisiae

PROVIDER: GSE125855 | GEO | 2019/01/30

REPOSITORIES: GEO

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Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation

Project description:Multiple DNA polymerases are needed to replicate genetic information. Here we describe the use of ribonucleotide incorporation as a biomarker of replication enzymology in vivo. We find that ribonucleotides are incorporated into the yeast nuclear genome in replicase specific and strand-specific patterns that identify replication origins and where polymerase switching occurs. Ribonucleotide density varies across the genome as a function of the replicase, base, local sequence and proximity to nucleosomes and transcription start sites. Ribonucleotides are present in one strand at high densityat mitochondrial replication origins, implying unidirectional replication of a circular genome. The evolutionary conservation of the enzymes that incorporate and process ribonucleotides in DNA suggests that the use of ribonucleotides as biomarkers of DNA synthesis in cells will have widespread applicability. Mapping genomic ribonucleotides in 14 Saccharomyces cerevisiae strains (seven DNA polymerase backgrounds, with or without RNH201), via HydEn-seq (end sequencing of genomic fragments generated by alkaline hydrolysis).

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Mapping polymerase usage by tracking ribonucleotide incorporation (HydEn-seq) during DNA replication

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Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA

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Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.

Project description:Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5' DNA end-mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5' DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.

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