Tan, Hwee Boon; (1993) Kinetic studies on the fidelity of DNA replication involving DNA templates containing O6-methylguanine. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Production by N-nitroso compounds of O6-alkylguanine (O6-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 O6>alkylG:T base-pairs is less stable than that containing O6-alkylG:C pairs. In the work presented in this thesis, the kinetics of incorporation by Klenow fragment of Escherichia coli DNA polymerase I of thymine (T), and of cytosine (C), opposite O6-meG in the template DNA strand were examined. Both T and C were incorporated opposite O6-meG much slower than nucleotides forming regular A:T or G:C base pairs. Using an excess of Klenow over DNA and various concentrations of dXTF and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed Klenow-DNA complex was measured (pre-steady state kinetics). The results were consistent with the kinetic scheme: 1. polymerase-DNA binds dNTP; 2. conformational change in polymerase; 3. formation of phosphodiester between the dNTP and the 3'-OH of primer; 4. conformational change of polymerase; 5. release of pyrophsphate. The results were analysed 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, kforward = 3.9 s-1 and kback = 1.9 s-1 for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-l). The equilibrium constants for each step suggest that the greatest change in the Gibbs' free energy occurs at the conformational change after polymerisation, and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic. The Kms calculated from the rate constants (Km = 33.5 μM (24.0-46.7)* for both dTTP and dCTP [* 5% and 95% confidence limits]) were close to the approximate Kms obtained from Michaelis-Menten analysis of the initial rates of pre-steady state polymerisation (Km, = 30-35 μM for T and C). The measured progress of independently determined steady state experiments (i.e. polymerisation under conditions of excess DNA over Klenow) was close to that predicted from these calculated rate constants. The incorporation of the nucleotide following C in an O6-meG:C pair was much slower than that following T in an O^-meGiT pair. Taken with the available structural data (Kalnik et al., 1988a, b), this suggests that the discrimination in favour of the incorporation of T opposite O6-meG arises mainly because the T:O6-meG base-pair retains the Watson-Crick configuration (with the N1 of the purine juxtaposed to N3 of the pyrimidine), whereas the C:O6-meG base-pair is a wobble base pair with a distorted phosphodiester link 3' to the C. The slow incorporation of C opposite O6-meG, and of the next correct nucleotide following the incorporation of C, can be ascribed to the stereochemical problems encountered when forming the distorted phosphodiester links. The recent X-ray crystallography data (Beese et al., 1993) of a Klenow complexed with duplex DNA provided evidence that Klenow fragment interacts with the primer-template through the phosphodiester backbone, thus an incorporation event that produces a distortion in the phosphodiester backbone, such as the incorporation of C opposite O6-meG, could very well reduce the rate of its incorporation.

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