Active Oxygen-induced DNA Damage and its Repair Mechanisms

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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