Abstract
We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondrial and cytosol.
Highlights
We examined the intracellular distribution of S-oxodGTPase (S-oxo-7,S-dihydrodeoxyguanosine triphosphatase) encoded by the MTHI gene, a human mutator homologue
The human 8-oxo-dGTPase shows a considerable degree of amino acid sequence homology with the E. coli MutT protein [13], and expression of the human cDNA in mutT-deficient E. coli cells efficiently reduced the increased frequency of A:T to C:G transversion to the level seen in wild type strain [14]
Several lines of evidence obtained in this study show that the mitochondrial matrix possesses 8-oxo-dGTPase and suggest that the 8-oxo-dGTPase present in mitochondria and cytosol fractions is the product of the same gene
Summary
8-oxoguanine, 8-oxo-7,8-dihydroguanine; 8-oxo-dGTPase, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase; APMSF, (p-amidinophenyl)methanesulfonyl fluoride hydrochloride; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography. The oxidized form of guanine is formed in the nucleotide pool of the cell and can be eliminated by the mutT gene product. The human 8-oxo-dGTPase shows a considerable degree of amino acid sequence homology with the E. coli MutT protein [13], and expression of the human cDNA in mutT-deficient E. coli cells efficiently reduced the increased frequency of A:T to C:G transversion to the level seen in wild type strain [14]. The repair of oxidized mitochondrial DNA and elimination of 8-oxo-dGTP from the. A system for repair of oxidatively damaged DNA in the mitochondria has been described [23] It is uncertain whether the mitochondria possess mechanismts) for eliminating 8-oxo-dGTP from dNTP pool. Several lines of evidence obtained in this study show that the mitochondrial matrix possesses 8-oxo-dGTPase and suggest that the 8-oxo-dGTPase present in mitochondria and cytosol fractions is the product of the same gene
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