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

Abstract

Ribonucleotides are frequently incorporated into DNA during eukaryotic replication. Here we map the 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 of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the role of DNA polymerases α and δ in 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, 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.