Kalam M Abul, Basu Ashis K
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.
Chem Res Toxicol. 2005 Aug;18(8):1187-92. doi: 10.1021/tx050119r.
Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T >> C > A > G, the same at the GUC site was T >> A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G(8-oxo)T 12-mer duplex with a deltadeltaG of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG(8-oxo)(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG(8-oxo)(AP*) sequence relative to (AP*)G(8-oxo)T. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG(8-oxo)(AP*) sequence context.
DNA 簇状损伤是电离辐射公认的特征。作为 DNA 同一条链上的一种簇状损伤模型，我们使用噬菌粒载体评估了在猴肾细胞中与尿嘧啶相邻的 8-氧代鸟嘌呤（8-oxoG）的诱变潜力。尿嘧啶残基在体内会被尿嘧啶 DNA 糖基化酶切除，产生一个无碱基位点（AP 位点）。在 GUGTC 或 GTGUC 序列背景下的单个尿嘧啶产生了超过 60%含有 GTGTC 的子代，这表明在 AP 位点或尿嘧啶相对处发生了 dAMP 掺入，但超过 30%的群体显示 U 分别被 A、C 或 G 取代，这表明在 AP 位点相对处也分别发生了 dTMP、dGMP 或 dCMP 掺入。虽然在 GUG 位点靶向碱基替换的偏好是 T >> C > A > G，但在 GUC 位点则是 T >> A > C > G。我们得出结论，AP 位点相对处的碱基掺入是序列依赖性的。对于 8-oxoG，与在 TG(8-oxo)T 序列背景下单一 8-oxoG 产生的 23 - 24%的 G-->T 突变体相比，串联损伤 UG(8-oxo)T 和 TG(8-oxo)U 分别产生了约 60%和超过 85%不包含 TGT 序列的子代。当尿嘧啶与 8-oxoG 相邻时，检测到相当一部分串联突变。我们发现最有趣的是，在 TG(8-oxo)U 位点包括单突变和串联突变的总靶向 G(8-oxo)-->T 颠换相对于 UG(8-oxo)T 位点的约 30%接近 60%。TG(8-oxo)U 序列处较高的突变频率可能源于更易出错的 DNA 聚合酶的变化。热变性实验表明，在 12 聚体的 TG(8-oxo)(AP*)序列中 8-oxoG:C 对的 Tm 低于(AP*)G(8-oxo)T 12 聚体中的，ΔΔG 为 0.8 kcal/mol（其中 AP代表四氢呋喃，即无碱基位点模型）。当将每个序列中的 8-oxoG:C 对与 8-oxoG:A 对进行比较时，发现前者比后者更稳定。对于ΔΔG 为 1.6 kcal/mol 的(AP)G(8-oxo)T 12 聚体双链体，8-oxoG 相对处 C 比 A 的偏好性在 TG(8-oxo)(AP*)12 聚体双链体中降至 0.4 kcal/mol。这表明相对于(AP*)G(8-oxo)T，在 TG(8-oxo)(AP*)序列中聚合酶区分掺入 dCMP 而非 dAMP 的效率会更低。此外，在 TG(8-oxo)(AP*)序列背景下，错配修复蛋白识别和切除 8-oxoG 相对处 A 的效率可能会受损。
