| Co-Investigator(Kenkyū-buntansha) |
SAKUMI Kunihiko Kyushu University, Medical Inst.Bioregulation, 生体防御医学研究所, 助手 (50211933)
NAKABEPPU Yusaku Kyushu University, Medical Inst.Bioregulation, 生体防御医学研究所, 助手 (30180350)
TSUZUKI Teruhisa kyushu University, Medical Inst.Bioregulation, 生体防御医学研究所, 助教授 (40155429)
DEMPLE Bruce Harvard University, School of Public Health, 公衆衛生学部, 教授
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
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
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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
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