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

By Marc Audebert, J. Pablo Radicella and Miral Dizdaroglu

Abstract

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (α-hOgg1-Ser326) (wild-type α-hOgg1). Mutant forms of hOgg1 with mutations Arg46→Gln (α-hOgg1-Gln46) and Arg154→His (α-hOgg1-His154) have previously been identified in human tumors. The mutant proteins α-hOgg1-Gln46 and α-hOgg1-His154 were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type α-hOgg1 were investigated using γ-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 γ-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 α-hOgg1 was greater than that by α-hOgg1-Gln46 and α-hOgg1-His154. 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 32P-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 γ-irradiated DNA. Wild-type α-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg154, in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type α-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms α-hOgg1-Gln46 and α-hOgg1-His154 found in human tumors are defective in their catalytic capacities

Topics: Article
Publisher: Oxford University Press
Year: 2000
OAI identifier: oai:pubmedcentral.nih.gov:102664
Provided by: PubMed Central
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