Effect of Single Mutations in the OGG1 Gene Found in Human Tumors on the Substrate Specificity of the OGG1 Protein
M Audebert, J. P. Radicella, M. Dizdaroglu
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 Arg-46->Gln (α-hOgg1-Gln46) and Arg-154->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 was 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 a-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 34-mer 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/Cytyt 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/Cytyt 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 clearly defective in their catalytic capacities.
Nucleic Acids Research
2, 6-Diamino-4-hydroxy-5-formamidopyrimid, 8-Hydroxguanine, DNA repair, Free radicals, mutations
, Radicella, J.
and Dizdaroglu, M.
Effect of Single Mutations in the OGG1 Gene Found in Human Tumors on the Substrate Specificity of the OGG1 Protein, Nucleic Acids Research
(Accessed November 30, 2023)