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

We have investigated the effect of single amino acid substitutions of conse rved arginines on the catalytic activities of the human Ogg1 protein (alpha -hOgg1-Ser(326)) (wild-type alpha-hOgg1). Mutant forms of hOgg1 with mutati ons Arg(46)-->Gln (alpha-hOgg1-Gln(46)) and Arg(154)-->His (alpha-hOgg1-His (154)) have previously been identified in human tumors. The mutant proteins alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these pro teins and wild-type alpha-hOgg1 were investigated using gamma-irradiated DN A and the technique of gas chromatography/isotope-dilution mass spectrometr y. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (F apyGua) and 8-hydroxyguanine (8-OH-Gua) from gamma-irradiated DNA containin g a multiplicity of base lesions. Michaelis-Menten kinetics of excision wer e measured. Significant differences between excision kinetics of these thre e enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type alph a-hOgg1 was greater than that by alpha-hOgg1-Gln(46) and alpha-hOgg1-His(15 4) The latter mutant protein was less active than the former. The diminishe d activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using P-32-labeled 34mer olig onucleotide duplexes containing a single 8-OH-Gua paired to each of the fou r DNA bases. The results obtained with the oligonucleotide containing the 8 -OH-Gua/Cyt pair were in good agreement with those observed with gamma-irra diated DNA. Wild-type alpha-hOgg1 and its mutants repaired the three mismat ches less efficiently than the 8-OH-Gua/ Cyt pair. The substitution of Arg( 154), in addition to diminishing the activity on 8-OH-Gua, relaxes the sele ctivity found in the wild-type alpha-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms alpha-hOgg1-Gln(46) a nd alpha-hOgg1-His(154) found in human tumors are defective in their cataly tic capacities.