Effects of N(2)-alkylguanine, O(6)-alkylguanine, and abasic lesions on DNA binding and bypass synthesis by the euryarchaeal B-family DNA polymerase vent (exo(-)).

Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo(-)) from the euryarchaeon Thermococcus litoralis was studied with three types of DNA lesions-N(2)-alkylG, O(6)-alkylG, and an abasic (AP) site-in comparison with a model Y-family pol Dpo4 from Sulfolobus solfataricus, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo(-)) readily bypassed N(2)-methyl(Me)G and O(6)-MeG, but was strongly blocked at O(6)-benzyl(Bz)G and N(2)-BzG, whereas Dpo4 efficiently bypassed N(2)-MeG and N(2)-BzG and partially bypassed O(6)-MeG and O(6)-BzG. Vent (exo(-)) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo(-)) showed ~110-, 180-, and 300-fold decreases in catalytic efficiency (k(cat)/K(m)) for nucleotide insertion opposite an AP site, N(2)-MeG, and O(6)-MeG but ~1800- and 5000-fold decreases opposite O(6)-BzG and N(2)-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ~13-fold decreases opposite N(2)-MeG and N(2)-BzG but ~260-370-fold decreases opposite O(6)-MeG, O(6)-BzG, and the AP site. Vent (exo(-)) preferentially misinserted G opposite N(2)-MeG, T opposite O(6)-MeG, and A opposite an AP site and N(2)-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo(-)) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo(-)) is as or more efficient as Dpo4 in synthesis opposite O(6)-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) N(2)-alkylGs than Vent (exo(-)), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo(-)) accepts nonbulky DNA lesions (e.g., N(2)- or O(6)-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove N(2)-alkylG lesions over major-groove or noninstructive lesions.