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Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

ABSTRACT: Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3-01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3-01/pol3-01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 x 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3-01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co-regulated with these genes. We propose that the POL3 3'-->5' exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ-->mutHLS pathway of DNA replication error correction in Escherichia coli.

SUBMITTER: Morrison A

PROVIDER: S-EPMC413358 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

Morrison A A Johnson A L AL Johnston L H LH Sugino A A

The EMBO journal 19930401 4

Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corres ...[more]

PMID: 8385605

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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.

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Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

Publications

Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

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