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
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
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DOI：
10.1021/bi026912q
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发表时间：
2003-03-04
期刊：
影响因子：
2.9
通讯作者：
Geacintov, NE
中科院分区：
文献类型：
--
作者：
Huang, XW;Kolbanovskiy, A;Geacintov, NE
体外检查了来自苯并[a]芘7,8-二醇9,10-环氧化物衍生物（(+)-7R,8S,9S,10R立体异构体）与N-2-鸟嘌呤（G*）结合的单个大块损伤侧翼碱基对Y家族聚合酶pol K（hDinB1）催化的跨损伤旁路的影响。病变位于六个不同的 43 聚体 5'-...XG*Y... 序列（X、Y = C、T 或 G，所有其他碱基保持固定）的中间附近。互补的 dCTP 优先插入所有序列中与 G* 相对的位置；然而，插入的其他 dNTP 的比例随着 X 和 Y 的函数而变化。XG*Y 中的 dCTP 插入效率 f(ins) = (V-max/K-m)(ins) 比 XGY 序列中的小，大约为 50-90（GG*T 和 GG*C）或 5000-25000（TG*G 和 CG*G）。值得注意的是，在 XG*Y 序列中，f(ins) 变化多达 3 个数量级，其中 3' 侧病变侧翼的 G 最小，5' 侧加合物侧翼的 G 最大。就在病灶之外的一步引物延伸效率 (f(ext)) 通常小于鳍，并且还取决于碱基序列。然而，在运行开始实验中，在所有序列中观察到（+）-陷阱-[BP]-N-2-dG 加合物的相当有效的跨损伤旁路，在 [dNTP] > K-m 的条件下观察到完全或接近完全的引物延伸。这里的关键特征是动力学参数 V-max 的相对稳健的值，在 (+)-陷阱-[BP]-N-2-dG 加合物的存在下，该值要么适度减小，甚至增强。与损伤对 V-max 的微小影响相比，XG*Y 中的表观 K-m 值比未修改的 XGY 序列大几个数量级。因此，在 [dNTP] < K-m 的条件下，绕过 (+)-traps-[BP]-N-2-dG 加合物是相当低效的。这些考虑因素在体内 [dNTP] 小于或等于 K-m 时可能很重要，并且 pol K 对 (+)-traps-[BP]-N-2-dG 的跨损伤旁路的效率可能明显低于体外较高 dNTP 浓度的效率。根据加合物的可能构象和旁路聚合酶的已知结构特征讨论了跨损伤旁路的碱基序列依赖性特征。
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.