Production by N-nitroso compounds of O-6-alkylguanine (O-6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O-6-alkylG:T base pairs is less stable than that containing O-6-alkylG:C pairs. We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O-6-MeG in the template DNA strand. Both T and C were incorporated opposite O-6-MeG much slower than nucleotides forming regular A:T or G:C base pairs. Using various concentrations of dTTP, dCTP, or their phosphorothioate (S-p)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics). The results were consistent with the kinetic scheme (Kuchta, R. D., Benkovic, P., and Benkovic, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate. The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, k(forward) = 3.9 s(-1) and k(back) = 1.9 s(-1); for dCTP, k(forward) = 0.7 s(-1) and k(back) = 0.9 S-1). These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4). The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic. K-m values obtained from Michaelis-Menten analysis of the initial rates of pre-steady-state polymerization were 27.6 and 26.4 mu M for T and C, respectively. These calculated rate constants closely predicted the progress of independently determined steady-state experiments (i.e. excess DNA over KF) and also predicted the measured K-m. The incorporation of the nucleotide following C in an O-6-MeG:C pair was much slower than that following T in an O-6-MeG:T pair. Structural data has shown that the T:O-6-alkylG base pair retains the Watson-Crick configuration (with N1 of the purine juxtaposed to N3 of the pyrimidine), whereas the C:O-6-allkylG base pair is a wobble base pair. The C:O-6-alkylG base pair has distorted phosphodiester links both 3' and 5' to the C (Kalnik, M. W., Li, B. F. L., Swann, P. F., and Patel, D. J. (1989) Biochemistry 28, 6170-6181 and 6182-6192). The slow incorporation of C opposite O-6-MeG and of the next correct nucleotide following the incorporation of C can be ascribed to the stereochemical problems encountered when forming these distorted phosphodiester links.
