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PMID: 9241232 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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.

Nucleic acids research ·Vol. 25 ·No. 16 ·1997-08-15 ·Pages 3204-11

Girard PM, Guibourt N, Boiteux S

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.

MeSH Terms
Binding Sites Borohydrides DNA Repair DNA-Binding Proteins/genetics DNA-Formamidopyrimidine Glycosylase Escherichia coli Proteins Fungal Proteins/genetics Genes, Fungal Lysine/chemistry N-Glycosyl Hydrolases/genetics Oxidation-Reduction Saccharomyces cerevisiae/genetics Structure-Activity Relationship Substrate Specificity
Chemicals
Borohydrides DNA-Binding Proteins Escherichia coli Proteins Fungal Proteins sodium borohydride N-Glycosyl Hydrolases DNA-Formamidopyrimidine Glycosylase DNA-formamidopyrimidine glycosylase, E coli Lysine
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Girard P M
Laboratoire de Radiobiologie du DNA, CEA/DSV, UMR217 Centre National de la Recherche Scientifique, Département de Radiobiologie et Radiopathologie, BP6, F-92265 Fontenay aux Roses, France.
Guibourt N
Boiteux S
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/25.16.3204
Published
1997-08-15
Pages
3204-11
Language
English
Region
England
NLM ID
0411011
PMCID
PMC146873
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/25.16.3204 PMC Full Text View journal details
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