We have examined the mechanism of DNA polymerase β (pol β) lesion discrimination using alkylated dNTP versus alkylated DNA template substrates and the pol β variants R253M and E249K. Both of these amino acid variants are located in the loop region of the palm domain and are known to play a role in pol β fidelity and discrimination of 3′-azido-3′- deoxythymidine triphosphate substrates. We observed that these variants affect O6-methyldeoxyguanosine- (m6G-) modified dNTP discrimination without affecting m6G template translesion synthesis. Under steady-state conditions, the ratio of inherent reactivity values for the modGTP substrate relative to the dGTP substrate was greater for both variant polymerases than for wild-type (WT) pol β. Biochemical assays of translesion synthesis using m6G lesion-containing templates demonstrated no significant differences between the variants and WT. Using N-methyl-N-nitrosourea- (MNU-) modified DNA templates in the HSV-tk in vitro assay, no difference among the enzymes in the frequency of alkylation-induced G to A transition mutations was observed. However, differences among the polymerases in the frequency of alkylation-induced C to A transversions were observed, consistent with a mutator tendency for E249K and an antimutator tendency for R253M. We conclude that a specific interaction at the loop of the palm domain is involved in pol β discrimination of the m6G lesion when present on the incoming dNTP substrate but not when present in the DNA template. Our data support a role for the flexible loop in pol β error discrimination.
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