PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of narLink to Publisher's site
 
Nucleic Acids Res. 1997 August 15; 25(16): 3204–3211.
PMCID: PMC146873

The Ogg1 protein of Saccharomyces cerevisiae: a 7,8-dihydro-8-oxoguanine DNA glycosylase/AP lyase whose lysine 241 is a critical residue for catalytic activity.

Abstract

The OGG1 gene of Saccharomyces cerevisiae codes for a DNA glycosylase that excises 7,8-dihydro-8- oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Fapy) from damaged DNA. In this paper, we have analysed the substrate specificity and the catalytic mechanism of the Ogg1 protein acting on DNA subtrates containing 8-OxoG residues or apurinic/apyrimidinic (AP) sites. The Ogg1 protein displays a marked preference for DNA duplexes containing 8-OxoG placed opposite a cytosine, the rank order for excision of 8-OxoG and cleavage efficiencies being 8-OxoG/C >8-OxoG/T >>8-OxoG/G and 8-OxoG/A. The cleavage of the DNA strand implies the excision of 8-OxoG followed by abeta-elimination reaction at the 3'-side of the resulting AP site. The Ogg1 protein efficiently cleaves a DNA duplex where a preformed AP site is placed opposite a cytosine (AP/C). In contrast, AP/T, AP/A or AP/G substrates are incised with a very low efficiency. Furthermore, cleavage of 8-OxoG/C or AP/C substrates implies the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre (NaBH4). Therefore, the Ogg1 protein is a eukaryotic DNA glycosylase/AP lyase. Sequence homology searches reveal that Ogg1 probably shares a common ancestor gene with the endonuclease III of Escherichia coli. A consensus sequence indicates a highly conserved lysine residue, K120 of endonuclease III or K241 of Ogg1, respectively. Mutations of K241 to Gln (K241Q) and Arg (K241R) have been obtained after site directed mutagenesis of OGG1. Mutation K241Q completely abolishes DNA glycosylase activity and covalent complex formation in the presence of NaBH4. However, the K241Q mutant still binds DNA duplexes containing 8-OxoG/C. In contrast, K241R mutation results in a catalytically active form of Ogg1. These results strongly suggest that the free amino group of Lys241 is involved in the catalytic mechanism of the Ogg1 protein.

Full Text

The Full Text of this article is available as a PDF (290K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Demple B, Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem. 1994;63:915–948. [PubMed]
  • Breimer LH. Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage. Mol Carcinog. 1990;3(4):188–197. [PubMed]
  • Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7915–7922. [PubMed]
  • Michaels ML, Miller JH. 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. [PMC free article] [PubMed]
  • Grollman AP, Moriya M. Mutagenesis by 8-oxoguanine: an enemy within. Trends Genet. 1993 Jul;9(7):246–249. [PubMed]
  • Boiteux S. Properties and biological functions of the NTH and FPG proteins of Escherichia coli: two DNA glycosylases that repair oxidative damage in DNA. J Photochem Photobiol B. 1993 Jul;19(2):87–96. [PubMed]
  • Michaels ML, Cruz C, Grollman AP, Miller JH. 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. [PubMed]
  • Duwat P, de Oliveira R, Ehrlich SD, Boiteux S. Repair of oxidative DNA damage in gram-positive bacteria: the Lactococcus lactis Fpg protein. Microbiology. 1995 Feb;141(Pt 2):411–417. [PubMed]
  • 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. [PubMed]
  • van der Kemp PA, Thomas D, Barbey R, de Oliveira R, Boiteux S. Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5197–5202. [PubMed]
  • Thomas D, Scot AD, Barbey R, Padula M, Boiteux S. Inactivation of OGG1 increases the incidence of G . C-->T . A transversions in Saccharomyces cerevisiae: evidence for endogenous oxidative damage to DNA in eukaryotic cells. Mol Gen Genet. 1997 Mar 26;254(2):171–178. [PubMed]
  • Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr 22;362(6422):709–715. [PubMed]
  • Chetsanga CJ, Lindahl T. Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli. Nucleic Acids Res. 1979 Aug 10;6(11):3673–3684. [PMC free article] [PubMed]
  • Boiteux S, O'Connor TR, Laval J. Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein. EMBO J. 1987 Oct;6(10):3177–3183. [PubMed]
  • Breimer LH. Enzymatic excision from gamma-irradiated polydeoxyribonucleotides of adenine residues whose imidazole rings have been ruptured. Nucleic Acids Res. 1984 Aug 24;12(16):6359–6367. [PMC free article] [PubMed]
  • Tchou J, Kasai H, Shibutani S, Chung MH, Laval J, Grollman AP, 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. [PubMed]
  • Boiteux S, Gajewski E, Laval J, Dizdaroglu M. Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization. Biochemistry. 1992 Jan 14;31(1):106–110. [PubMed]
  • Tchou J, Bodepudi V, Shibutani S, Antoshechkin I, Miller J, Grollman AP, Johnson F. Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA. J Biol Chem. 1994 May 27;269(21):15318–15324. [PubMed]
  • Bailly V, Verly WG, O'Connor T, Laval J. Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli [formamidopyrimidine]DNA glycosylase. Biochem J. 1989 Sep 1;262(2):581–589. [PubMed]
  • O'Connor TR, Laval J. Physical association of the 2,6-diamino-4-hydroxy-5N-formamidopyrimidine-DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5222–5226. [PubMed]
  • Graves RJ, Felzenszwalb I, Laval J, O'Connor TR. Excision of 5'-terminal deoxyribose phosphate from damaged DNA is catalyzed by the Fpg protein of Escherichia coli. J Biol Chem. 1992 Jul 15;267(20):14429–14435. [PubMed]
  • Bhagwat M, Gerlt JA. 3'- and 5'-strand cleavage reactions catalyzed by the Fpg protein from Escherichia coli occur via successive beta- and delta-elimination mechanisms, respectively. Biochemistry. 1996 Jan 16;35(2):659–665. [PubMed]
  • Nash HM, Bruner SD, Schärer OD, Kawate T, Addona TA, Spooner E, Lane WS, Verdine GL. Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily. Curr Biol. 1996 Aug 1;6(8):968–980. [PubMed]
  • Dodson ML, Michaels ML, Lloyd RS. Unified catalytic mechanism for DNA glycosylases. J Biol Chem. 1994 Dec 30;269(52):32709–32712. [PubMed]
  • Sun B, Latham KA, Dodson ML, Lloyd RS. Studies on the catalytic mechanism of five DNA glycosylases. Probing for enzyme-DNA imino intermediates. J Biol Chem. 1995 Aug 18;270(33):19501–19508. [PubMed]
  • Tchou J, Grollman AP. The catalytic mechanism of Fpg protein. Evidence for a Schiff base intermediate and amino terminus localization of the catalytic site. J Biol Chem. 1995 May 12;270(19):11671–11677. [PubMed]
  • Boiteux S, Huisman O. Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12. Mol Gen Genet. 1989 Jan;215(2):300–305. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Boiteux S, Belleney J, Roques BP, Laval J. Two rotameric forms of open ring 7-methylguanine are present in alkylated polynucleotides. Nucleic Acids Res. 1984 Jul 11;12(13):5429–5439. [PMC free article] [PubMed]
  • Castaing B, Geiger A, Seliger H, Nehls P, Laval J, Zelwer C, Boiteux S. Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins. Nucleic Acids Res. 1993 Jun 25;21(12):2899–2905. [PMC free article] [PubMed]
  • Ortigão JF, Rösch H, Selter H, Fröhlich A, Lorenz A, Montenarh M, Seliger H. Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation. Antisense Res Dev. 1992 Summer;2(2):129–146. [PubMed]
  • Castaing B, Boiteux S, Zelwer C. DNA containing a chemically reduced apurinic site is a high affinity ligand for the E. coli formamidopyrimidine-DNA glycosylase. Nucleic Acids Res. 1992 Feb 11;20(3):389–394. [PMC free article] [PubMed]
  • de Oliveira R, van der Kemp PA, Thomas D, Geiger A, Nehls P, Boiteux S. Formamidopyrimidine DNA glycosylase in the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1994 Sep 11;22(18):3760–3764. [PMC free article] [PubMed]
  • Schrock RD, 3rd, Lloyd RS. Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V. The position of the alpha NH2 moiety affects catalytic activity. J Biol Chem. 1993 Jan 15;268(2):880–886. [PubMed]
  • Piersen CE, Prince MA, Augustine ML, Dodson ML, Lloyd RS. Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate. J Biol Chem. 1995 Oct 6;270(40):23475–23484. [PubMed]
  • Thayer MM, Ahern H, Xing D, Cunningham RP, Tainer JA. Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure. EMBO J. 1995 Aug 15;14(16):4108–4120. [PubMed]
  • Eide L, Bjørås M, Pirovano M, Alseth I, Berdal KG, Seeberg E. Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10735–10740. [PubMed]
  • Roldán-Arjona T, Anselmino C, Lindahl T. Molecular cloning and functional analysis of a Schizosaccharomyces pombe homologue of Escherichia coli endonuclease III. Nucleic Acids Res. 1996 Sep 1;24(17):3307–3312. [PMC free article] [PubMed]
  • Lu R, Nash HM, Verdine GL. A mammalian DNA repair enzyme that excises oxidatively damaged guanines maps to a locus frequently lost in lung cancer. Curr Biol. 1997 Jun 1;7(6):397–407. [PubMed]

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