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Nat Struct Mol Biol. 2015 Mar;22(3):185-91. doi: 10.1038/nsmb.2957. Epub 2015 Jan 26.

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

Author information

1
Genome Integrity &Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institute of Health (NIH), Research Triangle Park, North Carolina, USA.
2
Integrative Bioinformatics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, USA.
3
Department of Genetics, High Throughput Sequencing Facility, University of North Carolina, Chapel Hill, North Carolina, USA.
4
Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York, USA.

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.

PMID:
25622295
PMCID:
PMC4351163
DOI:
10.1038/nsmb.2957
[Indexed for MEDLINE]
Free PMC Article

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