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

Anders R. Clausen, Scott A. Lujan, Adam B. Burkholder, Clinton D. Orebaugh, Jessica S. Williams, Maryam F. Clausen, Ewa P. Malc, Piotr A. Mieczkowski, David C. Fargo, Duncan J. Smith, Thomas A. Kunkel

Research output: Contribution to journalArticlepeer-review

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 and δin lagging-strand replication and of DNA polymerase ε 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.

Original languageEnglish (US)
Pages (from-to)185-191
Number of pages7
JournalNature Structural and Molecular Biology
Volume22
Issue number3
DOIs
StatePublished - Mar 6 2015

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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