Effects of <i>N</i>²‑Alkylguanine, <i>O</i>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 <i>Thermococcus litoralis</i> was studied with three types of DNA lesions<i>N</i>²-alkylG, <i>O</i>⁶-alkylG, and an abasic (AP) sitein comparison with a model Y-family pol Dpo4 from <i>Sulfolobus solfataricus</i>, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo^–) readily bypassed <i>N</i>²-methyl­(Me)­G and <i>O</i>⁶-MeG, but was strongly blocked at <i>O</i>⁶-benzyl­(Bz)­G and <i>N</i>²-BzG, whereas Dpo4 efficiently bypassed <i>N</i>²-MeG and <i>N</i>²-BzG and partially bypassed <i>O</i>⁶-MeG and <i>O</i>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 (<i>k</i>_cat/<i>K</i>_m) for nucleotide insertion opposite an AP site, <i>N</i>²-MeG, and <i>O</i>⁶-MeG but ∼1800- and 5000-fold decreases opposite <i>O</i>⁶-BzG and <i>N</i>²-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ∼13-fold decreases opposite <i>N</i>²-MeG and <i>N</i>²-BzG but ∼260–370-fold decreases opposite <i>O</i>⁶-MeG, <i>O</i>⁶-BzG, and the AP site. Vent (exo^–) preferentially misinserted G opposite <i>N</i>²-MeG, T opposite <i>O</i>⁶-MeG, and A opposite an AP site and <i>N</i>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 <i>O</i>⁶-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) <i>N</i>²-alkylGs than Vent (exo^–), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo^–) accepts nonbulky DNA lesions (e.g., <i>N</i>²- or <i>O</i>⁶-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove <i>N</i>²-alkylG lesions over major-groove or noninstructive lesions