Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 proteinand the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations

We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged b ases from DNA exposed to gamma-irradiation, Excision products were identifi ed and quantified using gas chromatography/isotope dilution mass spectromet ry (GC/IDMS), The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GS T-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4 -hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiat ion in a solution saturated with N2O or air, Fourteen other lesions, includ ing oxidised purines and pyrimidines, were not excised from these substrate s, Catalytic constants were measured for the excision of 8-OH-Gua and FapyG ua from DNA gamma-irradiated under N2O. The k(cat)/K-m values 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-alpha-hOgg1 protein were compared to that of a polymorph ic form of alpha-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 alpha-hOgg1-Ser(326) and mutant alpha-hOgg1-Cys(326) proteins . The GST-alpha-hOgg1-Cys(326) protein was purified and its substrate speci ficity was determined by GC/IDMS analysis. The results show that the GST-al pha-hOgg1-Cys(326) protein efficiently excises 8-OH-Gua and FapyGua from ga mma-irradiated DNA, The k(cat)/K-m values for excision of 8-OH-Gua and Fapy Gua were 2.82 x 10(-5) and 4.43 x 10(-5) (min(-1) nM(-1)), respectively. Fu rthermore, we compared the capacity of these two forms of alpha-hOgg1 to ac t on substrates containing 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimid ine (Me-FapyGua). The k(cat)/K-m values for excision of Me-FapyGua were 278 x 10(-5) and 319 x 10(-5) (min(-1) nM(-1)), respectively. Cleavage of 34me r oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apu rinic/apyrimidinic site paired with a cytosine was also investigated. The r esults show that both GST-alpha-hOgg1-Ser(326) and GST-alpha-hOgg1-Cys(326) catalyse the various cleavage reactions at very similar rates. Furthermore , both proteins efficiently complement the mutator phenotype of the fpg mut Y mutant of Escherichia coli.