Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan.
Chem Res Toxicol. 2012 Aug 20;25(8):1771-6. doi: 10.1021/tx300259x. Epub 2012 Jul 30.
The formation of 8-oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA and in the nucleotide pool results in G:C→T:A and A:T→C:G substitution mutations, respectively, since G(O) can pair with both C and A. In this study, the role of DNA polymerase κ in the mutagenicity of G(O) was investigated, using a supF shuttle plasmid propagated in human U2OS cells. This translesion synthesis DNA polymerase was knocked down by siRNA, and plasmid DNAs containing G(O):C and G(O):A pairs were transfected into the knock-down cells. The supF plasmid DNAs replicated in the cells were then introduced into Escherichia coli. Mutation analyses indicated that the knock-down of DNA polymerase κ by siRNA decreased the frequency of G:C→T:A mutation caused by G(O):C, although no effects of the DNA polymerase κ reduction were observed for the A:T→C:G substitution induced by G(O):A. These results suggested that DNA polymerase κ is involved in the mutagenic bypass of G(O) in living human cells, when the damaged base is generated by direct DNA oxidation.
8-氧代-7,8-二氢鸟嘌呤（G（O），8-羟基鸟嘌呤）在 DNA 中和核苷酸池中形成，分别导致 G:C→T:A 和 A:T→C:G 取代突变，因为 G（O）可以与 C 和 A 配对。在这项研究中，使用在人 U2OS 细胞中繁殖的 supF 穿梭质粒，研究了 DNA 聚合酶 κ 在 G（O）致突变性中的作用。通过 siRNA 敲低这种跨损伤合成 DNA 聚合酶，并将含有 G（O）：C 和 G（O）：A 对的质粒 DNA 转染到敲低细胞中。然后将在细胞中复制的 supF 质粒 DNA 导入大肠杆菌。突变分析表明，siRNA 敲低 DNA 聚合酶 κ 降低了由 G（O）：C 引起的 G:C→T:A 突变频率，尽管 G（O）：A 诱导的 A:T→C:G 取代没有观察到 DNA 聚合酶 κ 减少的影响。这些结果表明，当直接 DNA 氧化产生受损碱基时，DNA 聚合酶 κ 参与了活人体细胞中 G（O）的诱变旁路。
