Effects of base sequence context on translesion synthesis past a bulky (+)-trans-anti-B[a]P-N2-dG lesion catalyzed by the Y-family polymerase pol K

The effects of bases flanking single bulky lesions derived from the binding of a benzo[a]pyrene 7,8-diol 9,10-epoxide derivative ((+)-7R,8S,9S,10R stereoisomer) to N-2-guanine (G*) on translesion bypass catalyzed by the Y-family polymerase pol K (hDinB1) were examined in vitro. The lesions were positioned near the middle of six different 43-mer 5'-...XG*Y... sequences (X, Y = C, T, or G, with all other bases remaining fixed). The complementary dCTP is preferentially inserted opposite G* in all of the sequences; however, the proportions of other dNTPs inserted varies as a function of X and Y. The dCTP insertion efficiencies, f(ins) = (V-max/K-m)(ins) are smaller in the XG*Y than in XGY sequences by factors of similar to50-90 (GG*T and GG*C) or 5000-25000 (TG*G and CG*G). Remarkably, in XG*Y sequences, f(ins) varies by as much as 3 orders of magnitude, being smallest with G flanking the lesions on the 3'-side and highest with G flanking the adducts on the 5'-side. One-step primer extension efficiencies just beyond the lesions (f(ext)) are generally smaller than fins and also depend on base sequence. However, reasonably efficient translesion bypass of the (+)-traps-[BP]-N-2-dG adducts is observed in all sequences in running-start experiments with full, or nearly full, primer extension being observed under conditions of [dNTP] > K-m. The key features here are the relatively robust values of the kinetic parameters V-max that are either diminished to a moderate extent or even enhanced in the presence of the (+)-traps-[BP]-N-2-dG adducts. In contrast to the small effects of the lesions on V-max, the apparent K-m values are orders of magnitude greater in XG*Y than in the unmodified XGY sequences. Thus the bypass of (+)-traps-[BP]-N-2-dG adducts under conditions when [dNTP] < K-m is quite inefficient. These considerations may be of importance in vivo where [dNTP] less than or equal to K-m, and the translesion bypass of the (+)-traps-[BP]-N-2-dG by pol K may be significantly less efficient than in vitro at higher dNTP concentrations. The base sequence-dependent features of translesion bypass are discussed in terms of the possible conformations of the adducts and the known structural features of bypass polymerases.