DNA Polymerase ζ-Dependent Lesion Bypass in Saccharomyces cerevisiae Is Accompanied by Error-Prone Copying of Long Stretches of Adjacent DNA

Olga V. Kochenova, Danielle L. Daee, Tony M. Mertz, Polina V Shcherbakova

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

Original languageEnglish (US)
Article numbere1005110
JournalPLoS genetics
Volume11
Issue number3
DOIs
StatePublished - Mar 31 2015

Fingerprint

DNA-directed DNA polymerase
bypass
DNA-Directed DNA Polymerase
lesion
Saccharomyces cerevisiae
Mutation Rate
DNA
synthesis
mutation
Plasmids
Nucleotides
Mutation
Eukaryota
plasmids
assays
nucleotides
Chromosomes
plasmid
eukaryotic cells
assay

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Molecular Biology
  • Genetics
  • Genetics(clinical)
  • Cancer Research

Cite this

DNA Polymerase ζ-Dependent Lesion Bypass in Saccharomyces cerevisiae Is Accompanied by Error-Prone Copying of Long Stretches of Adjacent DNA. / Kochenova, Olga V.; Daee, Danielle L.; Mertz, Tony M.; Shcherbakova, Polina V.

In: PLoS genetics, Vol. 11, No. 3, e1005110, 31.03.2015.

Research output: Contribution to journalArticle

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