Dynamics of translesion DNA synthesis catalyzed by the bacteriophage T4 exonuclease-deficient DNA polymerase

The mechanism and dynamics of translesion DNA synthesis were evaluated usin g primer/templates containing a tetrahydrofuran moiety designed to mimic an abasic site. Steady-state kinetic analysis reveals that the T4 DNA polymer ase preferentially incorporates dATP across from the abasic site with 100-f old higher efficiency than the other nucleoside triphosphates. Under steady -state conditions, the catalytic efficiency of dATP incorporation across fr om an abasic site is only 220-fold lower than that across from T. Surprisin gly, misincorporation across from T is favored 4-6-fold versus replication across an abasic site, suggesting that the dynamics of the polymerization c ycle are differentially affected by formation of aberrant base pairs as opp osed to the lack of base-pairing capabilities afforded by the abasic site. Linear pre-steady-state time courses were obtained for the incorporation of any dNTP across from an abasic site, indicating that chemistry or a step p rior to chemistry is rate-limiting for the polymerization cycle. Low elemen tal effects (<3) measured by substituting the <alpha>-thiotriphosphate anal ogues for dATP, dCTP, and dGTP indicate that chemistry is not solely rate-l imiting. Single-turnover experiments yield k(pol)/K-d values that are essen tially identical to k(cat)/K-m values and provide further evidence that the conformational change preceding chemistry is rate-limiting. Extension beyo nd an A:abasic mispair is approximately 20-fold and 100-fold faster than ex tension beyond a G:abasic mispair or C:abasic mispair, respectively. Extens ion from the G:abasic or A:abasic site mispair generates significant elemen tal effects (between 5 and 20) and suggests that chemistry is at least part ially rate-limiting for extension beyond either mispair.