Mutational specificity of thymine nucleotide depletion in yeast

Relative to normal growth conditions for a wild-type strain of the yeast Saccharomyces cerevisiae, withholding thymidylate (dTMP) severely diminished the dTTP pool but elevated the dATP, dGTP and dCTP levels (120-, 8.5- and 3.6-fold, respectively) in an isogenic dTMP auxotroph. This treatment also increased the frequency of mutations in a tRNA gene (SUP4-o) by 15-fold. Single base-pair events accounted for 97% of the 89 SUP4-o mutations characterized by DNA sequencing and the ratio of transversions to transitions was 3-fold greater than that for spontaneous substitutions in the wild-type strain. This difference was due to decreases in the fractions of transitions and an increase in the proportion of A.T-->T.A transversions. The largest increases in mutation frequency were observed for transversions at A.T pairs, consistent with dATP and dGTP being incorporated in place of dTTP during DNA replication. Similarly, misinsertion of dATP and dGTP could have promoted the single base-pair deletions and insertion detected. Analysis of the distributions of substitutions indicated no preference for dATP misinsertion to occur at sites flanked by a specific 5' or 3' base or on the transcribed or nontranscribed strands. However, the presence of mutational hotspots and site-specific variations in the substitution frequencies implied a role for DNA sequence context in the mutational specificity of dTTP depletion.