Biochemical and physicochemical characterization of normal and variant forms of human MTH1 protein with antimutagenic activity

Hiroyuki Yakushiji, Fabrice Maraboeuf, Masayuki Takahashi, Zeng Sui Deng, Shun-Ichiro Kawabata, Yusaku Nakabeppu, Mutsuo Sekiguchi

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44 Citations (Scopus)

Abstract

8-0xo-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 an 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 α-helix content. Substitution of valine for methionine at the residue 83 of MYH1 protein appears to lead to alteration in the secondary structure which renders the protein more labile than the normal type protein.

Original languageEnglish
Pages (from-to)181-194
Number of pages14
JournalMutation Research - DNA Repair
Volume384
Issue number3
DOIs
Publication statusPublished - Sep 1 1997

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Toxicology
  • Genetics

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