USE OF SINGLE-TURNOVER KINETICS TO STUDY BULKY ADDUCT BYPASS BY T7-DNA POLYMERASE

The mechanism by which T7 DNA polymerase (exo-) by passes N-2-acetylam inofluorene (AAF) and N-2-aminofluorene (AF) adducts was studied by si ngle-turnover kinetics. These adducts are known to be mutagenic in sev eral cell types, and their bypass was studied in the framework of unde rstanding how they promote mutations. Synthetic primer/templates were made from a template sequence containing a single guanine, to which th e adducts were covalently attached, and one of three primers whose 3' ends were various distances from the adduct in the annealed substrates . Upon approaching the site of either adduct, the polymerase was found to add nucleotides as rapidly as to unmodified primer/templates, unti l just opposite the lesion. The incorporation rate of dCTP (at 100 muM ) opposite AF-dG or AAF-dG was approximately 5 x 10(4)- and 4 X 10(6)- fold slower, respectively, than incorporation at the same position int o an unmodified primer/template. The polymerase dissociated from the s ites of the adducts at approximately the same rate that it dissociated from unmodified DNA. Correct nucleotide incorporation was favored bot h opposite and immediately after AF-dG. However, at both positions, dA TP was the most rapidly misincorporated nucleotide. Misincorporation o f dATP was more rapid than correct nucleotide incorporation both oppos ite and immediately after AAF-dG. These results are discussed in terms of the effects of AF and AAF adducts in vivo.