Steady-state and pre-steady-state kinetic analysis of dNTP insertion opposite 8-oxo-7,8-dihydroguanine by Escherichia coli polymerases I exo super(-) and II exo super(-)

Escherichia coli polymerases (pol) I exo super(-) (KF super(-)) and pol II exo super(-) (pol II super(-)) were used as model enzymes with a DNA primer/template complex (12/16-mer) to examine the kinetics of incorporation of dCTP and dATP at the site of an 8-oxo-7,8-dihydroguanine (8-oxoGua) residue, compared to guanine (Gua). In steady-state assays (with DNA in excess) the rate of incorporation (k sub(cat)) was dCTP > dATP and the K sub(m,dATP) < K sub(m,dCTP) during incorporation opposite 8-oxoGua with both polymerases. Pre-steady-state kinetic curves (rapid-quench analysis) for the addition of C opposite 8-oxoGua or Gua by KF super(-) and pol II super(-) were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of A opposite 8-oxoGua did not display burst kinetics with either enzyme. Reduced rates of incorporation of the dCTP alpha S and dATP alpha S phosphorothioate analogs suggest that the rates of incorporation of A and C opposite 8-oxoGua are limited during polymerization by the rate of phosphodiester bond formation. Neither polymerase appears to discriminate between adducted and nonadducted DNA substrate for binding. Kinetic assays performed with varying dCTP concentrations indicate that dCTP has a higher K sub(d) and lower k sub(p) (polymerization rate) for incorporation opposite 8-oxoGua compared to Gua. Furthermore, the dATP binding affinities with KF super(-) and pol II super(-) were similar to 10- and similar to 3-fold lower, respectively, than that of dCTP as determined in competition assays with mixtures of dCTP and dATP. Microscopic rate constants were estimated by mathematical analysis of dNTP concentration dependence curves. Both polymerases preferentially extended the A:8-oxoGua pair while extension of the C:8-oxoGua pair was greatly impaired. Based on these findings, the fidelity of KF super(-) and pol II super(-) during replication of 8-oxoGua depends on contributions from nucleotide binding, the rate of phosphodiester bond formation, and the ease of base pair extension.