Abstract
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 kappa (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 approximately 50-90 (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 f(ins) and also depend on base sequence. However, reasonably efficient translesion bypass of the (+)-trans-[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 (+)-trans-[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 (+)-trans-[BP]-N(2)-dG adducts under conditions when [dNTP] < K(m) is quite inefficient. These considerations may be of importance in vivo where [dNTP] <or= K(m), and the translesion bypass of the (+)-trans-[BP]-N(2)-dG by pol kappa 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.
