Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W M644L and M644F mutants of yeast DNA polymerase ε

摘要

To better understand the functions and fidelity of DNA polymerase >ε (Pol >ε), we report here on the fidelity of yeast Pol >ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol >ε synthesize DNA with high fidelity, but M644F Pol >ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol >ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol >α (L868M/F) and Pol >δ (L612M), these data indicate that the active site location occupied by Met644 in Pol >ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol >ε is distinct from that of L868M/F Pol >α or L612M Pol >δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.