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

Research paper by Anders R AR Clausen, Scott A SA Lujan, Adam B AB Burkholder, Clinton D CD Orebaugh, Jessica S JS Williams, Maryam F MF Clausen, Ewa P EP Malc, Piotr A PA Mieczkowski, David C DC Fargo, Duncan J DJ Smith, Thomas A TA Kunkel

Indexed on: 27 Jan '15Published on: 27 Jan '15Published in: Nature Structural and Molecular Biology



Abstract

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.