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-)

Abstract

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, O6-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 O6-MeG, but was strongly blocked at O6-benzyl(Bz)G and N2-BzG, whereas Dpo4 efficiently bypassed N2-MeG and N2-BzG and partially bypassed O 6-MeG and O6-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 (kcat/Km) for nucleotide insertion opposite an AP site, N2-MeG, and O6-MeG but ∼1800- and 5000-fold decreases opposite O6-BzG and N2-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ∼13-fold decreases opposite N2-MeG and N2-BzG but ∼260-370-fold decreases opposite O6-MeG, O6-BzG, and the AP site. Vent (exo-) preferentially misinserted G opposite N2-MeG, T opposite O6-MeG, and A opposite an AP site and N2-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 O6-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) N2-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 O6-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove N2-alkylG lesions over major-groove or noninstructive lesions. © 2012 American Chemical Society.