Sulfolobus solfataricus DNA polymerase Dpo4 is partially inhibited by "wobble" pairing between O6-methylguanine and cytosine, but accurate bypass is preferred

We examined the effect of a single O-6-methylguanine (O-6-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O-6-MeG, with C being the major product (similar to 70%) and T or A being the minor species (similar to 20% or similar to 10%, respectively), consistent with steady- state kinetic parameters. Transient- state kinetic experiments revealed that k(pol), the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O-6-MeG was similar to 6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre- steady state. The lack of any structural information regarding how O-6-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O-6-MeG. A structure containing T opposite O-6-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher Km, dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O-6-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.