RESUMEN
Adenine paired with 8-hydroxyguanine (oh(8)G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells. Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His(6)-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh(8)G and A:G mismatches by DNA cleavage assay and gel mobility shift assay. We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q(324) and 2-Q(310) proteins with type 1-H(324) and 2-H(310) proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain. In a reaction buffer with a low salt (0-50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh(8)G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh(8)G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh(8)G, was proportionally reduced. The glycosylase activity on A:oh(8)G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins. These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh(8)G under physiological salt concentrations.
Asunto(s)
Adenina/metabolismo , Disparidad de Par Base/genética , Reparación del ADN/genética , ADN/metabolismo , Proteínas de Escherichia coli , Guanina/análogos & derivados , Guanina/metabolismo , N-Glicosil Hidrolasas/metabolismo , Animales , Secuencia de Bases , Liasas de Carbono-Oxígeno/química , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/aislamiento & purificación , Liasas de Carbono-Oxígeno/metabolismo , ADN/química , ADN/genética , ADN Glicosilasas , Reparación del ADN/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN-Formamidopirimidina Glicosilasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos/genética , Prueba de Complementación Genética , Humanos , Cinética , Mitocondrias/enzimología , Mutación/genética , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/aislamiento & purificación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Polimorfismo de Nucleótido Simple/genética , Cloruro de Potasio/farmacología , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Cloruro de Sodio/farmacología , SpodopteraRESUMEN
The human OGG1 gene encodes 8-hydroxyguanine DNA glycosylase. By RT-PCR analysis, five novel type 1 transcripts, in addition to eight known types (OGG1-types 1a to 1c and 2a to 2e), were identified. Among them, only the type 1a isoform contains both a nuclear localization signal and the entire DNA binding motif, suggesting the involvement of type 1a in chromosomal DNA repair. By Western blot analysis using a monoclonal antibody prepared by immunizing the whole type 1a protein, a 39 kDa type 1a protein was detected in lung cancer cell lines and peripheral lymphocytes. The type 1a protein was expressed at a similar level, irrespective of its polymorphic types characterized by distinct repair activity. By an immunocytochemical study, the majority of type 1a protein was localized in the nucleus. These results indicate that OGG1-type 1a protein is involved in the repair of 8-hydroxyguanine in chromosomal double-stranded DNA and constitutively expressed in cancerous and non-cancerous human cells.