Effect of Single Mutations in the OGG1 Gene Found in Human Tumors on the Substrate Specificity of the OGG1 Protein

Published

July 1, 2000

Author(s)

M Audebert, J. P. Radicella, M. 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 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.

Citation

Nucleic Acids Research

Volume

28

Issue

No. 14

Pub Type

Journals

Keywords

2, 6-Diamino-4-hydroxy-5-formamidopyrimid, 8-Hydroxguanine, DNA repair, Free radicals, mutations

Citation

Audebert, M. , Radicella, J. and Dizdaroglu, M. (2000), Effect of Single Mutations in the OGG1 Gene Found in Human Tumors on the Substrate Specificity of the OGG1 Protein, Nucleic Acids Research (Accessed May 10, 2024)