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

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 ?) 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 ? or a mutator Pol ? 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 rad14? strains have elevated mutation rates that depend on Pol ?. Rates are most strongly elevated for tandem base pair substitutions and clusters of multiple, closely spaced mutations. Both types of mutations depend on Pol ?, but not on Pol ?. Rates of each are further elevated in yeast strains bearing the rev3-979F allele. The results indicate that when Pol ? 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 ? to its evolutionary conservation and possibly to multistage carcinogenesis.

SUBMITTER: Stone JE

PROVIDER: S-EPMC3678557 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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

Stone Jana E JE Lujan Scott A SA Kunkel Thomas A TA Kunkel Thomas A TA

Environmental and molecular mutagenesis 20120911 9

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 muta ...[more]

PMID: 22965922

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Project description:Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase ?-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In this study, we examined how carboxymethylated DNA lesions, which included N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA), N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O(4)-carboxymethylthymidine (O(4)-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase ? (pol ?). Our results from steady-state kinetic assay showed that pol ? could readily bypass and extend past N(6)-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5'-nucleoside with high efficiency. By contrast, the polymerase could bypass N(4)-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol ? could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol ? experienced great difficulty in bypassing O(4)-CMdT and N3-CMdT, and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors.

Dataset Information

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

Publications

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

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