In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta

Several amino acids in the active site of family A DNA polymerases contribu te to accurate DNA synthesis. For two of these residues, family B DNA polym erases have conserved tyrosine residues in regions II and III that are sugg ested to have similar functions. Here we replaced each tyrosine with alanin e in the catalytic subunits of yeast DNA polymerases alpha, delta, epsilon, and zeta and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region It in Pol delta or Pol epsilon are inviable. Strains with same substitution in Rev3, the catal ytic subunit of Pol zeta, are nearly UV immutable, suggesting severe loss o f function. A strain with this substitution in Pol alpha (pol1-Y869A) is vi able, but it exhibits slow growth, sensitivity to hydroxyurea, and a sponta neous mutator phenotype for frameshifts and base substitutions. The pol1-Y8 69A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is cri tical for the function of all four eukaryotic family B DNA polymerases. Str ains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Pol alpha, delta, or -epsilon are viable and a strain with the homol ogous substitution in Rev3 is UV mutable. The Pola mutant has no obvious ph enotype. The Pol epsilon (pol.2-17831A) mutant is slightly sensitive to hyd roxyurea and is a semidominant mutator for spontaneous base substitutions a nd frameshifts. The Pol delta mutant (Pol3-Y708A) grows slowly, is sensitiv e to hydroxy-urea and methyl methanesulfonate, and is a strong base substit ution and frameshift, mutator. The pol3-Y708A/pol3-Y708A diploid grows slow ly and aberrantly. Mutation rates in the Pol alpha, -delta, and -epsilon mu tant strains are increased in a locus-specific manner by inactivation of PM S1-dependent DNA mismatch repair, suggesting that the mutator effects are d ue to reduced fidelity of chromosomal DNA replication. This could result di rectly from relaxed base selectivity of the mutant polymerases due to the a mino acid changes in the polymerase active site. In addition, the alanine s ubstitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation th at the pol3-Y708A mutation is recessive and its mutator effect is partially , suppressed by disruption of the REV3 gene.