The polymerase eta translesion synthesis DNA polymerase acts independently of the mismatch repair system to limit mutagenesis caused by 7,8-dihydro-8-oxoguanine in yeast

Abstract

Reactive oxygen species are ubiquitous mutagens that have been linked to both disease and aging. The most studied oxidative lesion is 7,8-dihydro-8-oxoguanine (GO), which is often miscoded during DNA replication, resulting specifically in GC --> TA transversions. In yeast, the mismatch repair (MMR) system repairs GO.A mismatches generated during DNA replication, and the polymerase eta (Poleta) translesion synthesis DNA polymerase additionally promotes error-free bypass of GO lesions. It has been suggested that Poleta limits GO-associated mutagenesis exclusively through its participation in the filling of MMR-generated gaps that contain GO lesions. In the experiments reported here, the SUP4-o forward-mutation assay was used to monitor GC --> TA mutation rates in strains defective in MMR (Msh2 or Msh6) and/or in Poleta activity. The results clearly demonstrate that Poleta can function independently of the MMR system to prevent GO-associated mutations, presumably through preferential insertion of cytosine opposite replication-blocking GO lesions. Furthermore, the Poleta-dependent bypass of GO lesions is more efficient on the lagging strand of replication and requires an interaction with proliferating cell nuclear antigen. These studies establish a new paradigm for the prevention of GO-associated mutagenesis in eukaryotes.

FIG. 1.

Mutation spectra of hbn1::SUP4-oF strains. The transcribed sequence of SUP4-o is shown in the 3′-to-5′ orientation from left to right. Base substitutions are indicated below the sequence of SUP4-o. G → T transversions are shaded in blue, and C → A transversions are shaded in yellow. The numbers above the sequence are given to indicate the relative position of the GC base pair, e.g., “4” indicates the fourth GC base pair in the sequence. + indicates an insertion event, Δ indicates a deletion event, and cdel indicates a complex deletion, in which a deletion is associated with a base substitution event.

FIG. 2.

Locations of SUP4-o alleles relative to ARS306. (A and B) In the hbn1::SUP4-o strains, SUP4-o is on the left side of ARS306, with the replication fork moving from right to left. (A) In the hbn1::SUP4-oF allele, the transcribed strand is the lagging-strand template and is indicated by a gray box. Sequencing of the transcribed strand in hbn1::SUP4-oF mutants uncovered lagging-strand G → T mutations and leading-strand C > A mutations (indicated in boldface). (B) In the hbn1::SUP4-oR allele, the transcribed strand is on the opposite DNA strand and is the leading-strand template. Sequencing of the transcribed strand in hbn1::SUP4-oR mutants uncovered leading-strand G → T mutations and lagging-strand C > A mutations. (C and D) In the agp1::SUP4-o strains, SUP4-o is on the right side of ARS306, with the replication fork moving from left to right. (C) In the agp1::SUP4-oF allele, the transcribed strand is the leading-strand template. Sequencing of the transcribed strand in agp1::SUP4-oF mutants uncovered leading-strand G → T mutations and lagging-strand C > A mutations. (D) In the agp1::SUP4-oR allele, the transcribed strand is on the opposite strand and is the lagging-strand template. Sequencing of the transcribed strand in agp1::SUP4-oR mutants uncovered lagging-strand G → T mutations and leading-strand C > A mutations. LD indicates leading-strand mutations, and LG indicates lagging-strand mutations.

FIG. 3.

Site-specific analysis of MMR and Polη activity in hbn1::SUP4-o strains. The rates of GC → TA transversions on the lagging strand (G → T in the hbn1::SUP4-oF strain and C > A in the hbn1::SUP4-oR strain) are shown on a log scale. Each site is labeled according to its position in the SUP4-o gene, i.e., 3G is the third guanine in the gene. Only those sites where at least five G → T or five C > A mutations were observed in two different strain backgrounds are included. An asterisk indicates that the mutation rate was calculated assuming one event, as none were detected at these specific sites.

FIG. 4.

Roles of MMR and Polη activities in preventing GO-associated mutagenesis.