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 κ

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 N²-guanine (G*) on translesion bypass catalyzed by the Y-family polymerase pol κ (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 ∼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]-N²-dG adducts is observed in all sequences in runningstart 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]-N²-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]-N²-dG adducts under conditions when [dNTP] < K_m is quite inefficient. These considerations may be of importance in vivo where [dNTP] < K_m, and the translesion bypass of the (+)-trans-[BP]-N²-dG by pol κ 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.