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Excision of Oxidatively Damaged DNA Bases by the Human Α-hOgg1 Protein and the Polymorphic Α-hOgg1 (Ser326Cys) Protein Which is Frequently Found in Human Populations



C. Dherin, J. P. Radicella, M. Dizdaroglu, S Boiteux


We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as α-hOgg1, for excision of damaged bases from DNA exposed to γ-irradiation. Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS). the GST-α-hOgg1 protein used in this study is a fusion of α-hOgg1 to the C-terminus of the GST protein. The results show that the GST-α-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to γ-irradiation in a solution saturated with N2O or air. Fourteen other lesions, including oxidized purines and pyrimidines, were not excised from these substrates. Catalytic constants were measured for the excision for 8-OH-Gua and FapyGua from DNA γ-irradiated under N2O. the Kcat/KMvalues for excision of 8-OH-Gua and FapyGua were 4.47 x 10-5 and 8.97 x 10-5 (min-1 nM-1), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-α-hOgg1 protein were compared to that of a polymorphic form of α-hOgg1 harbouring a Ser > Cys mutation at codon 326. In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type α-hOgg1-Ser326 and the mutant α-hOgg1-Cys326 proteins. The GST-α-αhOgg1-Cys326 protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-α-hOgg1-Cys326 protein efficiently excises 8-OH-Gua and FapyGua from γ-irradiated DNA. The kcat/Km values for excision of 8-OH-Gua and FapyGua were 2.82 x 10-5 and 4.43 x 10-5(min-1 nM-1), respectively. Furthermore, we compared the capacity of these two forms of α-hOgg1 to act on substrates containing 2,6-diamino-4 hydroxy-5-N-methylformamidopyrimidine (Me-FapyGua). The kcat/Kmvalues for excision of Me-FapyGua were 278 x 10-5 and 319 x 10-5 (min-1 nM-1), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-αhOgg1-Ser326 and GST-α-hogg1-Cys326 catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator pheno-type of the fpg mutY mutant of Escherichia coli.
Nucleic Acids Research
No. 20


8-hydroxyguanine, base excision repair, DNA glycosyloases, mutations, oxidative DNA damage, substrate specificity


Dherin, C. , Radicella, J. , Dizdaroglu, M. and Boiteux, S. (1999), Excision of Oxidatively Damaged DNA Bases by the Human Α-hOgg1 Protein and the Polymorphic Α-hOgg1 (Ser326Cys) Protein Which is Frequently Found in Human Populations, Nucleic Acids Research (Accessed April 15, 2024)
Created December 1, 1999, Updated February 17, 2017