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

The mechanism and dynamics of translesion DNA synthesis were evaluated using primer/templates containing a tetrahydrofuran moiety designed to mimic an abasic site. Steady-state kinetic analysis reveals that the T4 DNA polymerase preferentially incorporates dATP across from the abasic site with 100-fold higher efficiency than the other nucleoside triphosphates. Under steady-state conditions, the catalytic efficiency of dATP incorporation across from an abasic site is only 220-fold lower than that across from T. Surprisingly, misincorporation across from T is favored 4-6-fold versus replication across an abasic site, suggesting that the dynamics of the polymerization cycle are differentially affected by formation of aberrant base pairs as opposed 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 prior to chemistry is rate-limiting for the polymerization cycle. Low elemental effects (<3) measured by substituting the alpha-thiotriphosphate analogues for dATP, dCTP, and dGTP indicate that chemistry is not solely rate-limiting. Single-turnover experiments yield kpol/Kd values that are essentially identical to kcat/Km values and provide further evidence that the conformational change preceding chemistry is rate-limiting. Extension beyond an A:abasic mispair is approximately 20-fold and 100-fold faster than extension beyond a G:abasic mispair or C:abasic mispair, respectively. Extension from the G:abasic or A:abasic site mispair generates significant elemental effects (between 5 and 20) and suggests that chemistry is at least partially rate-limiting for extension beyond either mispair.