Biochemical and physicochemical characterization of normal and variant forms of human MTH1 protein with antimutagenic activity.
8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during cellular metabolism, and its misincorporation into DNA causes mutation. Human cells possess an enzyme that hydrolyzes 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing misincorporation of 8-oxo-7,8-dihydroguanine into DNA. Sequence analyses of the MTH1 gene, encoding the 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphatase (8-oxo-dGTPase) protein in human cell lines revealed that a G to A base substitution frequently occurs at codon 83, which causes a change of valine to methionine in the MTH1 protein [Wu, C. et al., Biochem. Biophys. Res. Commun. 214 (1995) 1239-1245]. Here we isolated cDNAs for the two types of MTH1 protein and expressed them in Escherichia coli mutT-. cells, devoid of their own 8-oxo-dGTPase activity. The two forms of proteins were purified to physical homogeneity, and amino acid analyses confirmed that the variant protein, Met83-MTH1, indeed carries the corresponding amino acid substitution. Met83-MTH1, but not normal type Val83-MTH1, was separated into two peaks in hydrophobic interacting chromatography. 8-Oxo-dGTPase activity of Met83-MTH1 is more thermolabile than that of Val83-MTH1. Circular dichroism (CD) and fluorescence spectroscopic analyses confirmed this conclusion. CD further indicated that Met83-MTH1 has a higher alpha-helix content.
Yakushiji
H
H
Department of Biochemistry, Kyushu University, Fukuoka, Japan.
Maraboeuf
F
F
Takahashi
M
M
Deng
Z S
ZS
Kawabata
S
S
Nakabeppu
Y
Y
Sekiguchi
M
M
D019943:Amino Acid Substitution / Q000235:genetics; D016587:Antimutagenic Agents / Q000737:chemistry* / Q000302:isolation & purification; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D045643:DNA Repair Enzymes; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D014644:Genetic Variation; D006358:Hot Temperature; D006801:Humans; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D016587:Antimutagenic Agents; D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; C078207:8-oxodGTPase; D045643:DNA Repair Enzymes