KINETIC-ANALYSIS OF THE CODING PROPERTIES OF O-6-METHYLGUANINE IN DNA- THE CRUCIAL ROLE OF THE CONFORMATION OF THE PHOSPHODIESTER BOND

Production by N-nitroso compounds of O-6-alkylguanine (O-6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, le ading to G:C to A:T transition mutations, despite the fact that DNA co ntaining 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 K lenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T ) and of cytosine (C) opposite O-6-MeG in the template DNA strand. Bot h T and C were incorporated opposite O-6-MeG much slower than nucleoti des forming regular A:T or G:C base pairs. Using various concentration s of dTTP, dCTP, or their phosphorothioate (S-p)-dNTP alpha S analogue s, or a mixture of dTTP and dCTP, the progress of incorporation of a s ingle nucleotide in a single catalytic cycle of a preformed KF-DNA com plex was measured (pre-steady-state kinetics). The results were consis tent with the kinetic scheme (Kuchta, R. D., Benkovic, P., and Benkovi c, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to po lymerase-DNA; (2) conformational change in polymerase; (3) formation o f phosphodiester between the dNTP and the 3'-OH of the primer; (4) con formational change of polymerase; (5) release of pyrophosphate. The re sults 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 r ates 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 bipha sic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the impor tance of the conformational change after polymerization by showing tha t the curves represented the sum of the rapid accumulation of the prod uct of step 3 followed by the slow conversion of that to the product o f step 5 (because of the rapidity of the release of pyrophosphate ther e was no significant accumulation of the product of step 4). The equil ibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymeriz ation and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic. K-m values obt ained from Michaelis-Menten analysis of the initial rates of pre-stead y-state polymerization were 27.6 and 26.4 mu M for T and C, respective ly. These calculated rate constants closely predicted the progress of independently determined steady-state experiments (i.e. excess DNA ove r KF) and also predicted the measured K-m. The incorporation of the nu cleotide following C in an O-6-MeG:C pair was much slower than that fo llowing 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-all kylG base pair is a wobble base pair. The C:O-6-alkylG base pair has d istorted 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, 6 170-6181 and 6182-6192). The slow incorporation of C opposite O-6-MeG and of the next correct nucleotide following the incorporation of C ca n be ascribed to the stereochemical problems encountered when forming these distorted phosphodiester links.