Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Mar 15;25(6):1170–1176. doi: 10.1093/nar/25.6.1170

Significance of the conserved amino acid sequence for human MTH1 protein with antimutator activity.

J P Cai 1, H Kawate 1, K Ihara 1, H Yakushiji 1, Y Nakabeppu 1, T Tsuzuki 1, M Sekiguchi 1
PMCID: PMC146569  PMID: 9092626

Abstract

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during normal cellular metabolism, and incorporation into DNA causes transversion mutation. Organisms possess an enzyme, 8-oxo-dGTPase, which catalyzes the hydrolysis of 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing the occurrence of mutation. There are highly conserved amino acid sequences in prokaryotic and eukaryotic proteins containing this and related enzyme activities. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site- directed mutagenesis of the cloned cDNA for human 8-oxo-dGTPase, and the activity and stability of mutant forms of the enzyme were examined. When lysine-38 was replaced by other amino acids, all of the mutants isolated carried the 8-oxo-dGTPase-negative phenotype. 8-Oxo-dGTPase-positive revertants, isolated from one of the negative mutants, carried the codon for lysine. Using the same procedure, the analysis was extended to other residues within the conserved sequence. At the glutamic acid-43, arginine-51 and glutamic acid-52 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild type protein. We propose that Lys-38, Glu-43, Arg-51 and Glu-52 residues in the conserved region are essential to exert 8-oxo-dGTPase activity.

Full Text

The Full Text of this article is available as a PDF (159.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abeygunawardana C., Weber D. J., Gittis A. G., Frick D. N., Lin J., Miller A. F., Bessman M. J., Mildvan A. S. Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase. Biochemistry. 1995 Nov 21;34(46):14997–15005. doi: 10.1021/bi00046a006. [DOI] [PubMed] [Google Scholar]
  2. Akiyama M., Horiuchi T., Sekiguchi M. Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion. Mol Gen Genet. 1987 Jan;206(1):9–16. doi: 10.1007/BF00326530. [DOI] [PubMed] [Google Scholar]
  3. Ames B. N., Gold L. S. Endogenous mutagens and the causes of aging and cancer. Mutat Res. 1991 Sep-Oct;250(1-2):3–16. doi: 10.1016/0027-5107(91)90157-j. [DOI] [PubMed] [Google Scholar]
  4. Bessho T., Tano K., Kasai H., Nishimura S. Deficiency of 8-hydroxyguanine DNA endonuclease activity and accumulation of the 8-hydroxyguanine in mutator mutant (mutM) of Escherichia coli. Biochem Biophys Res Commun. 1992 Oct 15;188(1):372–378. doi: 10.1016/0006-291x(92)92395-e. [DOI] [PubMed] [Google Scholar]
  5. Bessho T., Tano K., Kasai H., Ohtsuka E., Nishimura S. Evidence for two DNA repair enzymes for 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in human cells. J Biol Chem. 1993 Sep 15;268(26):19416–19421. [PubMed] [Google Scholar]
  6. Cai J. P., Kakuma T., Tsuzuki T., Sekiguchi M. cDNA and genomic sequences for rat 8-oxo-dGTPase that prevents occurrence of spontaneous mutations due to oxidation of guanine nucleotides. Carcinogenesis. 1995 Oct;16(10):2343–2350. doi: 10.1093/carcin/16.10.2343. [DOI] [PubMed] [Google Scholar]
  7. Cheng K. C., Cahill D. S., Kasai H., Nishimura S., Loeb L. A. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions. J Biol Chem. 1992 Jan 5;267(1):166–172. [PubMed] [Google Scholar]
  8. Cupples C. G., Miller J. H. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5345–5349. doi: 10.1073/pnas.86.14.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Demple B., Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem. 1994;63:915–948. doi: 10.1146/annurev.bi.63.070194.004411. [DOI] [PubMed] [Google Scholar]
  10. Frick D. N., Weber D. J., Abeygunawardana C., Gittis A. G., Bessman M. J., Mildvan A. S. NMR studies of the conformations and location of nucleotides bound to the Escherichia coli MutT enzyme. Biochemistry. 1995 Apr 25;34(16):5577–5586. doi: 10.1021/bi00016a032. [DOI] [PubMed] [Google Scholar]
  11. Furuichi M., Yoshida M. C., Oda H., Tajiri T., Nakabeppu Y., Tsuzuki T., Sekiguchi M. Genomic structure and chromosome location of the human mutT homologue gene MTH1 encoding 8-oxo-dGTPase for prevention of A:T to C:G transversion. Genomics. 1994 Dec;24(3):485–490. doi: 10.1006/geno.1994.1657. [DOI] [PubMed] [Google Scholar]
  12. Gajewski E., Rao G., Nackerdien Z., Dizdaroglu M. Modification of DNA bases in mammalian chromatin by radiation-generated free radicals. Biochemistry. 1990 Aug 28;29(34):7876–7882. doi: 10.1021/bi00486a014. [DOI] [PubMed] [Google Scholar]
  13. Kakuma T., Nishida J., Tsuzuki T., Sekiguchi M. Mouse MTH1 protein with 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphatase activity that prevents transversion mutation. cDNA cloning and tissue distribution. J Biol Chem. 1995 Oct 27;270(43):25942–25948. doi: 10.1074/jbc.270.43.25942. [DOI] [PubMed] [Google Scholar]
  14. Kamath A. V., Yanofsky C. Sequence and characterization of mutT from Proteus vulgaris. Gene. 1993 Nov 30;134(1):99–102. doi: 10.1016/0378-1119(93)90180-b. [DOI] [PubMed] [Google Scholar]
  15. Kang D., Nishida J., Iyama A., Nakabeppu Y., Furuichi M., Fujiwara T., Sekiguchi M., Takeshige K. Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria. J Biol Chem. 1995 Jun 16;270(24):14659–14665. doi: 10.1074/jbc.270.24.14659. [DOI] [PubMed] [Google Scholar]
  16. Kasai H., Crain P. F., Kuchino Y., Nishimura S., Ootsuyama A., Tanooka H. Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis. 1986 Nov;7(11):1849–1851. doi: 10.1093/carcin/7.11.1849. [DOI] [PubMed] [Google Scholar]
  17. Lin J., Abeygunawardana C., Frick D. N., Bessman M. J., Mildvan A. S. The role of Glu 57 in the mechanism of the Escherichia coli MutT enzyme by mutagenesis and heteronuclear NMR. Biochemistry. 1996 May 28;35(21):6715–6726. doi: 10.1021/bi953071x. [DOI] [PubMed] [Google Scholar]
  18. Maki H., Sekiguchi M. MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. Nature. 1992 Jan 16;355(6357):273–275. doi: 10.1038/355273a0. [DOI] [PubMed] [Google Scholar]
  19. McGoldrick J. P., Yeh Y. C., Solomon M., Essigmann J. M., Lu A. L. Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein. Mol Cell Biol. 1995 Feb;15(2):989–996. doi: 10.1128/mcb.15.2.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Michaels M. L., Cruz C., Grollman A. P., Miller J. H. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7022–7025. doi: 10.1073/pnas.89.15.7022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Michaels M. L., Miller J. H. The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). J Bacteriol. 1992 Oct;174(20):6321–6325. doi: 10.1128/jb.174.20.6321-6325.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mo J. Y., Maki H., Sekiguchi M. Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):11021–11025. doi: 10.1073/pnas.89.22.11021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moriya M., Ou C., Bodepudi V., Johnson F., Takeshita M., Grollman A. P. Site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in E. coli. Mutat Res. 1991 May;254(3):281–288. doi: 10.1016/0921-8777(91)90067-y. [DOI] [PubMed] [Google Scholar]
  24. Méjean V., Salles C., Bullions L. C., Bessman M. J., Claverys J. P. Characterization of the mutX gene of Streptococcus pneumoniae as a homologue of Escherichia coli mutT, and tentative definition of a catalytic domain of the dGTP pyrophosphohydrolases. Mol Microbiol. 1994 Jan;11(2):323–330. doi: 10.1111/j.1365-2958.1994.tb00312.x. [DOI] [PubMed] [Google Scholar]
  25. Nakabeppu Y., Oda S., Sekiguchi M. Proliferative activation of quiescent Rat-1A cells by delta FosB. Mol Cell Biol. 1993 Jul;13(7):4157–4166. doi: 10.1128/mcb.13.7.4157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nghiem Y., Cabrera M., Cupples C. G., Miller J. H. The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2709–2713. doi: 10.1073/pnas.85.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sakumi K., Furuichi M., Tsuzuki T., Kakuma T., Kawabata S., Maki H., Sekiguchi M. Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis. J Biol Chem. 1993 Nov 5;268(31):23524–23530. [PubMed] [Google Scholar]
  28. Sekiguchi M. MutT-related error avoidance mechanism for DNA synthesis. Genes Cells. 1996 Feb;1(2):139–145. doi: 10.1046/j.1365-2443.1996.d01-232.x. [DOI] [PubMed] [Google Scholar]
  29. Shibutani S., Takeshita M., Grollman A. P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature. 1991 Jan 31;349(6308):431–434. doi: 10.1038/349431a0. [DOI] [PubMed] [Google Scholar]
  30. Shigenaga M. K., Gimeno C. J., Ames B. N. Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9697–9701. doi: 10.1073/pnas.86.24.9697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tajiri T., Maki H., Sekiguchi M. Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli. Mutat Res. 1995 May;336(3):257–267. doi: 10.1016/0921-8777(94)00062-b. [DOI] [PubMed] [Google Scholar]
  32. Tchou J., Kasai H., Shibutani S., Chung M. H., Laval J., Grollman A. P., Nishimura S. 8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4690–4694. doi: 10.1073/pnas.88.11.4690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Treffers H. P., Spinelli V., Belser N. O. A Factor (or Mutator Gene) Influencing Mutation Rates in Escherichia Coli. Proc Natl Acad Sci U S A. 1954 Nov;40(11):1064–1071. doi: 10.1073/pnas.40.11.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wood M. L., Dizdaroglu M., Gajewski E., Essigmann J. M. Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry. 1990 Jul 31;29(30):7024–7032. doi: 10.1021/bi00482a011. [DOI] [PubMed] [Google Scholar]
  35. Wu C., Nagasaki H., Maruyama K., Nakabeppu Y., Sekiguchi M., Yuasa Y. Polymorphisms and probable lack of mutation in a human mutT homolog, hMTH1, in hereditary nonpoliposis colorectal cancer. Biochem Biophys Res Commun. 1995 Sep 25;214(3):1239–1245. doi: 10.1006/bbrc.1995.2419. [DOI] [PubMed] [Google Scholar]
  36. Yanofsky C., Cox E. C., Horn V. The unusual mutagenic specificity of an E. Coli mutator gene. Proc Natl Acad Sci U S A. 1966 Feb;55(2):274–281. doi: 10.1073/pnas.55.2.274. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES