Mutational specificity of DNA precursor pool imbalances in yeast arising from deoxycytidylate deaminase deficiency or treatment with thymidylate.

Disruption of the dCMP deaminase (DCD1) gene, or provision of excess dTMP to a nucleotide-permeable strain, produced dramatic increases in the dCTP or dTTP pools, respectively, in growing cells of the yeast Saccharomyces cerevisiae. The mutation rate of the SUP4-o gene was enhanced 2-fold by the dCTP imbalance and 104-fold by the dTTP imbalance. 407 SUP4-o mutations that arose under these conditions, and 334 spontaneous mutations recovered in an isogenic strain having balanced DNA precursor levels, were characterized by DNA sequencing and the resulting mutational spectra were compared. Significantly more (greater than 98%) of the changes resulting from nucleotide pool imbalance were single base-pair events, the majority of which could have been due to misinsertion of the nucleotides present in excess. Unexpectedly, expanding the dCTP pool did not increase the fraction of A.T----G.C transitions relative to the spontaneous value nor did enlarging the dTTP pool enhance the proportion of G.C----A.T transitions. Instead, the elevated levels of dCTP or dTTP were associated primarily with increases in the fractions of G.C----C.G or A.T----T.A. transversions, respectively. Furthermore, T----C, and possibly A----C, events occurred preferentially in the dcd1 strain at sites where dCTP was to be inserted next. C----T and A----T events were induced most often by dTMP treatment at sites where the next correct nucleotide was dTTP or dGTP (dGTP levels were also elevated by dTMP treatment). Finally, misinsertion of dCTP or dTTP did not exhibit a strand bias. Collectively, our data suggest that increased levels of dCTP and dTTP induced mutations in yeast via nucleotide misinsertion and inhibition of proofreading but indicate that other factors must also be involved. We consider several possibilities, including potential roles for the regulation and specificity of proofreading and for mismatch correction.

Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae.

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)