MUTATIONAL SPECIFICITY OF THYMINE NUCLEOTIDE DEPLETION IN YEAST

Relative to normal growth conditions for a wild-type strain of the yea st Saccharomyces cerevisiae, withholding thymidylate (dTMP) severely d iminished the dTTP pool but elevated the dATP, dGTP and dCTP levels (1 20-, 8.5- and 3.6-fold, respectively) in an isogenic dTMP auxotroph. T his 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 spontane ous substitutions in the wild-type strain. This difference was due to decreases in the fractions of transitions and an increase in the propo rtion of A . T --> T . A transversions. The largest increases in mutat ion frequency were observed for transversions at A . T pairs, consiste nt with dATP and dGTP being incorporated in place of dTTP during DNA r eplication. Similarly, misinsertion of dATP and dGTP could have promot ed the single base-pair deletions and insertion detected. Analysis of the distributions of substitutions indicated no preference for dATP mi sinsertion to occur at sites flanked by a specific 5' or 3' base or on the transcribed or nontranscribed strands. However, the presence of m utational hotspots and site-specific variations in the substitution fr equencies implied a role for DNA sequence context in the mutational sp ecificity of dTTP depletion.