Hashiguchi K, Zhang Q M, Sugiyama H, Ikeda S, Yonei S
Laboratory for Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyoku, Kyoto 606-8502, Japan.
Int J Radiat Biol. 2002 Jul;78(7):585-92. doi: 10.1080/09553000210130560.
2-Hydroxyadenine (2-ohA) is an oxidation product of adenine generated in DNA by ionizing radiation and various chemical oxidants. 2-ohA has mutational potential comparable to that of 8-oxoguanine in bacteria and mammalian cells. Recent studies have shown that 2-ohA is removed from DNA by a human MutY homolog, MYH protein, in vitro. On the other hand, the repair mechanisms for 2-ohA in Escherichia coli are not yet understood.
Gel shift assays were used to assess the binding activity of E. coli full-length MutY protein and its N-terminal (residues 1-226) domain (M25) to 2-ohA/G-, 2-ohA/A-, 2-ohA/C- and 2-ohA/T-containing 24-mer oligonucleotides. Furthermore, whether these proteins specifically cleave 2-ohA-containing duplex oligonucleotides was examined.
The purified MutY and M25 proteins had similar binding affinities to 2-ohA/G-, 2-ohA/A- and 2-ohA/C-containing oligonucleotides. MutY protein removed 2-ohA preferentially from 2-ohA/G mispairs. M25 protein showed the reduced catalytic activity for 2-ohA/G-containing oligonucleotides.
E. coli MutY protein has a DNA glycosylase activity that removes 2-ohA from 2-ohA/G mispairs in DNA. The C-terminal domain is required for the removal of 2-ohA from DNA, but is not crucial for binding to 2-ohA-containing oligonucleotides.
2-羟基腺嘌呤（2-ohA）是腺嘌呤在DNA中经电离辐射和各种化学氧化剂作用产生的氧化产物。在细菌和哺乳动物细胞中，2-ohA具有与8-氧代鸟嘌呤相当的致突变潜力。最近的研究表明，人MutY同源蛋白MYH可在体外从DNA中去除2-ohA。另一方面，大肠杆菌中2-ohA的修复机制尚不清楚。
采用凝胶迁移实验评估大肠杆菌全长MutY蛋白及其N端（第1至226位氨基酸）结构域（M25）与含2-ohA/G-、2-ohA/A-、2-ohA/C-和2-ohA/T的24聚体寡核苷酸的结合活性。此外，还检测了这些蛋白是否能特异性切割含2-ohA的双链寡核苷酸。
纯化的MutY和M25蛋白对含2-ohA/G-、2-ohA/A-和2-ohA/C的寡核苷酸具有相似的结合亲和力。MutY蛋白优先从2-ohA/G错配中去除2-ohA。M25蛋白对含2-ohA/G的寡核苷酸的催化活性降低。
大肠杆菌MutY蛋白具有DNA糖基化酶活性，可从DNA中的2-ohA/G错配中去除2-ohA。C端结构域是从DNA中去除2-ohA所必需的，但对与含2-ohA的寡核苷酸结合并不关键。
