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PMID: 10908322 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Effect of single mutations in the OGG1 gene found in human tumors on the substrate specificity of the Ogg1 protein.

Nucleic acids research ·Vol. 28 ·No. 14 ·2000-07-15 ·Pages 2672-8

Audebert M, Radicella JP, Dizdaroglu M

Abstract

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (alpha-hOgg1-Ser(326)) (wild-type alpha-hOgg1). Mutant forms of hOgg1 with mutations Arg(46)-->Gln (alpha-hOgg1-Gln(46)) and Arg(154)-->His (alpha-hOgg1-His(154)) have previously been identified in human tumors. The mutant proteins alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type alpha-hOgg1 were investigated using gamma-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from gamma-irradiated DNA containing a multiplicity of base lesions. Michaelis-Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type alpha-hOgg1 was greater than that by alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154). The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using (32)P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with gamma-irradiated DNA. Wild-type alpha-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg(154), in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type alpha-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) found in human tumors are defective in their catalytic capacities.

MeSH Terms
DNA/chemistry,genetics,metabolism DNA Repair DNA-Formamidopyrimidine Glycosylase Escherichia coli Proteins Guanine/analogs & derivatives,metabolism Humans Kinetics Mutation N-Glycosyl Hydrolases/genetics,metabolism Neoplasms/enzymology Pyrimidines/metabolism Substrate Specificity
Chemicals
Escherichia coli Proteins Pyrimidines 2,6-diamino-4-hydroxy-5-formamidopyrimidine 8-hydroxyguanine Guanine DNA N-Glycosyl Hydrolases DNA-Formamidopyrimidine Glycosylase DNA-formamidopyrimidine glycosylase, E coli
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Audebert M
CEA, Département de Radiobiologie et Radiopathologie, UMR217 CNRS-CEA, 60 avenue du Général Leclerc, 92265-Fontenay aux Roses, France.
Radicella J P
Dizdaroglu M
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/28.14.2672
Published
2000-07-15
Pages
2672-8
Language
English
Region
England
NLM ID
0411011
PMCID
PMC102664
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/28.14.2672 PMC Full Text View journal details
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