Effects of N-2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) adducts positioned in the active site of replicative polymerases on nucleotide and polymerase binding

DNA polymerases are able to attain an extraordinarily high fidelity of nucleotide incorporation by combining at least two approaches to distinguish between a correct versus incorrect nucleotide. First, non-complementary nucleotides bind to the polymerase in the ground state (open conformation) more weakly than a complementary dNTP. Second, it is believed that only a complementary nucleotide allows a conformational change to form a ternary complex (closed conformation) that aligns the nucleotide and 3'-OH of the primer in the correct orientation for the formation of phosphodiester bond. In the present work 28-mer AAF and AF-modified templates were constructed and were annealed with 22-mer primers such that the primer end one nucleotide before the adduct site. A gelshift analysis was used to determine how carcinogenic DNA adducts, N-acetyl-2-aminofluorene-dG (AAF-dG) and 2-aminofluorene-dG (AF-dG), effect dNTP binding to DNA polymerase I (Klenow fragment). Using this method it was found out that correct nucleotide (dCTP) has a dissociation constant, Kd, 11 &mgr;M for binding to the polymerase on an unmodified primer-template. When the template is modified with an AAF adduct situated in the polymerase active site, the kd increased to 68 &mgr;M indicating a weaker binding to the polymerase. The presence of an AF adduct effect the binding of dCTP to a lesser extent as compared to the AAF-modified case. In the presence of incorrect nucleotide, the nucleotide binding affinity decreased by 5-6 fold for unmodified and AF-modfied templates, whereas, the AFF adduct caused complete inhibition of nucleotide binding to the DNA-polymerase complex. The results are consistent with the crystal structure of T7 DNA polymerase bound to AAF and AF-modified primer-templates. These structures indicate that the AAF adduct intercalates behind the O helix of the finger subdomain pushing it into the active the active center and occluding the dNTP binding site of the polymerase. The AF adduct is not visible but clearly in a different location compared with AAF case.