Nomoto Minoru, Yamaguchi Raizo, Kohno Kimitoshi, Kasai Hiroshi
Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Japan, Yahata Nishi-Ku, Kitakyushu 807-8555, Japan.
Oncogene. 2002 Mar 7;21(11):1649-57. doi: 10.1038/sj.onc.1205237.
The distribution of oxidative damage to bases such as 8-hydroxyguanine (8-OH-Gua), was determined at the nucleotide level of resolution using the ligation-mediated PCR technique. Administration of a renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and subsequent formation of 8-OH-Gua in the kidney. Whole genomic DNA was isolated from the rat kidney with or without Fe-NTA treatment and then digested with formamidopyrimidine-DNA glycosylase (Fpg). As a target, we focused on the gene of a DNA repair enzyme for thymine glycol, Nth 1. Cleaved signals were found in exon 1 and exon 3, but not exon 5. Nucleosomes in these regions, enriched in damaged nucleotides, were highly accessible to micrococcal nuclease, especially in the kidney. Taking into account the function of the protein segment encoded by these regions, we discussed the molecular mechanism of the restricted formation of the damaged nucleotides.
使用连接介导的聚合酶链反应（PCR）技术，在核苷酸分辨率水平上测定了诸如8-羟基鸟嘌呤（8-OH-Gua）等碱基的氧化损伤分布。已知给予肾致癌物次氮基三乙酸铁（Fe-NTA）会诱导氧化应激，并随后在肾脏中形成8-OH-Gua。从经或未经Fe-NTA处理的大鼠肾脏中分离出全基因组DNA，然后用甲酰胺嘧啶-DNA糖基化酶（Fpg）进行消化。作为研究对象，我们重点关注了胸腺嘧啶乙二醇DNA修复酶Nth 1的基因。在第1外显子和第3外显子中发现了切割信号，但在第5外显子中未发现。这些区域富含受损核苷酸的核小体对微球菌核酸酶具有高度可及性，尤其是在肾脏中。考虑到这些区域编码的蛋白质片段的功能，我们讨论了受损核苷酸受限形成的分子机制。
