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. 2012 Dec;53(9):777-86.
doi: 10.1002/em.21728. Epub 2012 Sep 11.

DNA polymerase zeta generates clustered mutations during bypass of endogenous DNA lesions in Saccharomyces cerevisiae

Jana E Stone  1 , Scott A Lujan, Thomas A Kunkel, Thomas A Kunkel
Affiliations

DNA polymerase zeta generates clustered mutations during bypass of endogenous DNA lesions in Saccharomyces cerevisiae

Jana E Stone et al. Environ Mol Mutagen. 2012 Dec.

Abstract

Multiple sequence changes that are simultaneously introduced in a single DNA transaction have a higher probability of altering gene function than do single base substitutions. DNA polymerase zeta (Pol ζ) has been shown to introduce such clustered mutations under specific selective and/or DNA damage-producing conditions. In this study, a forward mutation assay was used to determine the specificity of spontaneous mutations generated in Saccharomyces cerevisiae when either wild-type Pol ζ or a mutator Pol ζ variant (rev3-L979F) bypasses endogenous lesions. Mutagenesis in strains proficient for nucleotide excision repair (NER) was compared to mutagenesis in NER-deficient strains that retain unrepaired endogenous DNA lesions in the genome. Compared to NER-proficient strains, NER-deficient rad14Δ strains have elevated mutation rates that depend on Pol ζ. Rates are most strongly elevated for tandem base pair substitutions and clusters of multiple, closely spaced mutations. Both types of mutations depend on Pol ζ, but not on Pol η. Rates of each are further elevated in yeast strains bearing the rev3-979F allele. The results indicate that when Pol ζ performs mutagenic bypass of endogenous, helix-distorting lesions, it catalyzes a short track of processive, error-prone synthesis. We discuss the implications of this unique catalytic property of Pol ζ to its evolutionary conservation and possibly to multistage carcinogenesis.

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Figures

Fig. 1
Fig. 1
Overall spontaneous mutation rates. Mutation rates are shown for (A) ura3 and (B) can1 forward mutation reporters. Mutation rates from the URA3 Orientation 1 strain and the isogenic Orientation 2 strain are shown in white and gray, respectively. Error bars indicate 95% confidence intervals.
Fig. 2
Fig. 2
Rates for each mutation type. Mutation spectra were determined by sequencing independently isolated 5-FOAR derivatives from each indicated genotype. The numbers in parenthesis under the genotypes indicate the total number of ura3 mutations examined for URA3 Orientation 1 and Orientation 2, respectively. Mutation rates are shown for (A) transitions, (B) transversions, (C) −1 frameshifts, (D) TBPS, and (E) clustered mutations (multiple mutations with <10 base pairs between each adjacent mutation). Genotypes are indicated at the bottom. Mutation rates from the URA3 Orientation 1 strain and the isogenic Orientation 2 strain are shown in white and gray, respectively. Mutation rates and error bars for each mutation type were calculated as indicated in the Materials and Methods. One-tailed Fisher exact tests were used to compare the number of mutations of a particular type to all other mutations in a pair of strains. Asterisks (*) indicate that a statistically significant increase is observed in the rev3-L979F strain when compared to the isogenic REV3 strain using the same test (P value = 0.0027). Plus signs (+) indicate that a statistically significant increase is observed for the rad14Δ strain when compared to the isogenic RAD14 strain (P values ≤ 0.0006).
Fig. 3
Fig. 3
Spectra of clustered and TBPS mutations observed in wild-type, rev3-L979F, rad14Δ, and rad14Δ rev3-L979F strains. The sequence of the URA3 coding sequence is shown in black text with dots marking every 10 bases. Mutations from the URA3 Orientation 1 strain are shown above the URA3 reference sequence and mutations from the isogenic URA3 Orientation 2 strain are shown below. Boxed letters indicate TBPS with the mutant sequence indicated within the box. Filled boxes indicate clustered mutations with the length of the box extending from the first to the last mutated base in the mutation cluster. Blue boxes indicate cbps, clustered mutations composed of only base substitutions; green boxes indicate clustered mutations that include ≥1 insertions; and red boxes indicate clustered mutations that include ≥1 deletions. A white number within a box indicates the number of identical clustered mutations observed. Mutation spectra for the entire URA3 ORF and the sequences of clustered mutations are shown in the Supporting Information.
Fig. 4
Fig. 4
Spectra of clustered and TBPS mutations observed in rad14Δ rad30Δ and rad14Δ rad30Δ rev3-L979F strains. All text and symbol are as designated in Figure 3. Red boxes and text indicate TBPS mutations considered to be at hotspots. Mutation spectra for the entire URA3 ORF and the sequences of clustered mutations are shown in the Supporting Information.
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