Effects of N2-Alkylguanine, O6-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 ...
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-N2-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 N2-methyl(Me)G, O6-MeG, and (partially) O6-benzyl(Bz)G but was blocked at N2-BzG, whereas Dpo4 effeiciently bypassed N2-MeG and N2-BzG and partially bypassed O6-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 ~1,800- and 5,000-fold decreases opposite O6-BzG and N2-BzG, respectively, compared to G, whereas Dpo4 showed little or only ~13-fold decreases opposite N2-MeG and N2-BzG but ~260- to 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 non-bulky DNA lesions (e.g. N2- 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 non-instructive lesions.



Nucleotide Analogs / Template Lesions


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