Active Oxygen-induced DNA Damage and its Repair Mechanisms

8-氧代-7，8-二氢-2 '-脱氧鸟苷5'-三磷酸（8-oxo-dGTP）在正常细胞代谢过程中在细胞的核苷酸库中形成，并且当其掺入DNA中时引起突变。以人MTH 1（Escherichia coli mut T homolog）cDNA为探针，克隆了小鼠8-oxo-dGT 3的cDNA，测序结果表明，小鼠MTH 1蛋白（分子量为17，896）由156个氨基酸残基组成。当小鼠8-氧代-dGT 3的cDNA在缺乏其自身8-氧代-dGT 3活性的大肠杆菌mutT突变体细胞中表达时，形成了与抗人MTH 1抗体交叉反应的18-kDa蛋白。在这样的细胞中，自发突变频率水平升高后恢复正常。在肝脏、胸腺和大肠中发现了高水平的8-氧代-dGT酶活性，而所有其他检查的器官中含有较少量的8-氧代-dGT酶活性。 ...更多信息 e酶。在胚胎干细胞中，存在极高水平的酶。本研究还涉及8-oxo-dGTP在人类细胞的核苷酸库中的产生和降解。(1)8-oxo-dGTP不仅可以通过直接氧化dGTP生成，还可以通过核苷二磷酸激酶磷酸化8-oxodGDP生成。(2)8-oxo-dGTP通过细胞8-oxo-dGTP酶活性快速降解为8-oxo-dGMP。这样产生的8-氧代-dGMP不能再磷酸化;鸟苷酸激酶将GMP和dGMP磷酸化为相应的核苷二磷酸，对8-氧代-dGMP完全无活性。(3)8-氧代-dGMP被核苷酸酶进一步降解为8-氧代-脱氧鸟苷。从人Jurkat细胞提取物中部分纯化了该酶，并阐明了作用模式。8-oxo-dGMP是该酶最优选的底物，其它核苷单磷酸以显著较低的速率裂解：8-oxo-dGMP的Km比dGMP低10倍，dGMP是该酶的第二最佳底物。该酶似乎将积累在细胞核苷酸库中的8-氧代-dGMP转化为易于分泌到细胞外部的形式。少

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is formed in the cnucleotide pool of a cell during normal cellular metabolism, and when it is incorporated into DNA causes mutation. Organisms possess 8-oxo-dGTPase, an enzyme that specifically degrades 8-oxo-dGTP to 8-oxo-dGMP.We isolated cDNA for mouse 8-oxo-dGTPase, using as a probe human MHTl (Escherichia coli mut T homolog) cDNA.The nucleotide sequence of the cDNA revealed thet the mouse MTHl protein (molecular weight of 17,896) comprises 156 amino acid residues. When the cDNA for mouse 8-oxo-dGTPase was expressed in E.coli mutT mutant cells devoid of their own 8-oxo-dGTPase activity, an 18-kDa protein, which is cross-reactive with an anti-human MTHl antibody, was formed. In such cells, the level of spontaneous mutation frequency the was elevated reverted to normal. High levels of 8-oxo-dGTPase activity were found in liver, thymus, and large intestine, whereas all other organs examined contained smaller amounts of th … More e enzyme. In embryonic stem cells, an exceedingly high level of the enzyme was present.The present study also deals with generation and degradation of 8-oxo-dGTP in the nucleotide pool of human cells. (1) 8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxodGDP by nucleoside diphosphate kinase. (2) 8-Oxo-dGTP is repidly degraded to 8-oxo-dGMP by cellular 8-oxo-dGTPase activity. 8-Oxo-dGMP thus produced cannot be rephosphorylated ; guanylate kinase, which phosphorylates both GMP and dGMP to the corresponding nucleoside diphosphates, is totally inactive for 8-oxo-dGMP.(3) 8-Oxo-dGMP is further degraded to 8-oxo-deoxyguanosine by a nucleotidase. The enzyme was partially purified from an extract of human Jurkat cells, and the mode of action was elucidated. 8-Oxo-dGMP is the most preferred substrate of the enzyme, and other nucleoside monophosphates are cleaved at significantly lowre rates : Km for 8-oxo-dGMP is 10 times lower than that for dGMP,the second best substrate for the enzyme. The enzyme appears to convert 8-oxo-dGMP,which accumulates in the cellular nucleotide pool, to a form readily excretable to the cell exterior. Less