Terato Hiroaki, Masaoka Aya, Asagoshi Kenjiro, Honsho Akiko, Ohyama Yoshihiko, Suzuki Toshinori, Yamada Masaki, Makino Keisuke, Yamamoto Kazuo, Ide Hiroshi
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Nucleic Acids Res. 2002 Nov 15;30(22):4975-84. doi: 10.1093/nar/gkf630.
Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (V(max)/K(m)) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [(3)H]Xan from the substrate. The treatment of E.coli with N-methyl-N'-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA(+) but not alkA(-) strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan.
一氧化氮（NO）和亚硝酸等氮氧化物对DNA中的鸟嘌呤进行亚硝化会导致黄嘌呤（Xan）和氧化鸟嘌呤（Oxa）的形成，这些损伤可能具有细胞毒性和致突变性。在本研究中，我们检测了大肠杆菌DNA N-糖基化酶对Xan和Oxa的修复能力。使用含有Xan和Oxa的特定底物进行切口测定表明，在测试的酶中，AlkA[与核酸内切酶（Endo）IV结合]和Endo VIII能够识别Xan。AlkA对Xan的活性（V(max)/K(m)）比对7-甲基鸟嘌呤的活性低5倍，Endo VIII对Xan的活性比对胸腺嘧啶二醇的活性低50倍。从底物中释放[(3)H]Xan进一步证实了AlkA和Endo VIII对Xan的活性。用N-甲基-N'-硝基-N-亚硝基胍处理大肠杆菌会增加alkA(+)菌株而非alkA(-)菌株细胞提取物中的Xan切除活性。相对于野生型菌株，alkA和nei（Endo VIII基因）双突变体而非单突变体对亚硝酸表现出更高的敏感性。AlkA和Endo VIII对Oxa也表现出切除活性，但该活性远低于对Xan的活性。
