On the mechanism of preferential incorporation of dAMP at abasic sites in translesional DNA synthesis. Role of proof reading activity of DNA polymerase and thermodynamic characterization of model template-primers containing an abasic site

Abstract

DNA polymerases preferentially incorporate dAMP opposite abasic sites (A-rule). The mechanism of the A-rule can be studied by analyzing three dissected stages of the reaction including (i) initial nucleotide insertion, (ii) proofreading excision of the inserted nucleotide and (iii) extension of the nascent primer terminus. To assess the role of the stage (ii) in the A-rule, kinetic parameters of the proofreading excision of primer terminus nucleotides opposite abasic sites were determined using E.coli DNA polymerase I Klenow fragment. The relative efficiency of the excision (V_max/K_m) revealed that removal of A was the least favored of the four nucleotides, but the differences in the efficiencies between excision of A and the other nucleotides was less than 2-fold. In addition, in an attempt to reconcile kinetic data associated with the stages (i) or (ii), the differences in free energy changes (ΔΔG°) for the formation of model template-primer termini containing XN pairs (X = abasic site, N = A, G, C or T) were determined by temperature dependent UV-melting measurements. The order of ΔΔG° was XG > XA = XC ≥ XT, with ΔΔG° being 0.5 kca/mol for the most stable XG and the least stable XT. Based on these data, the role of the stage (ii) and energetic aspects of the A-rule are discussed.