RESUMEN
The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1. Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired with a cytosine, [8-OxoG.C], are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1) nM(-)(1) for the wild-type and the K241R protein, respectively. On the other hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities of yOgg1. In contrast, the K241R and K241Q proteins have conserved wild-type DNA binding properties. K(dapp) values for binding of [8-OxoG.C] are 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respectively. The results also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins. Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand. On the complementary strand, only the cytosine placed opposite Pr was strongly protected. The same footprints were observed with the K241R and K241Q proteins, confirming their wild-type DNA binding properties. These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizable substrates such as 8-OxoG or an AP site.
Asunto(s)
Arginina/genética , Proteínas de Unión al ADN/química , Glutamina/genética , Lisina/genética , N-Glicosil Hidrolasas/química , Saccharomyces cerevisiae/enzimología , Sitios de Unión/efectos de los fármacos , Catálisis , Ciclopentanos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , ADN-Formamidopirimidina Glicosilasa , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Furanos/farmacología , Guanosina/análogos & derivados , Guanosina/metabolismo , Hidrólisis , Radical Hidroxilo/análisis , Radical Hidroxilo/metabolismo , Cinética , N-Glicosil Hidrolasas/antagonistas & inhibidores , N-Glicosil Hidrolasas/aislamiento & purificación , N-Glicosil Hidrolasas/metabolismo , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Glicoles de Propileno/farmacología , Purinas/metabolismo , Pirimidinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
A spontaneous mutator strain of Escherichia coli (fpg mutY) was used to clone the OGG1 gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7,8-dihydro-8-oxoguanine (8-OxoG). E. coli (fpg mutY) was transformed by a yeast DNA library, and clones that showed a reduced spontaneous mutagenesis were selected. The antimutator activity was associated with pYSB10, an 11-kbp recombinant plasmid. Cell-free extracts of E. coli (fpg mutY) harboring pYSB10 possess an enzymatic activity that cleaves a 34-mer oligonucleotide containing a single 8-oxoG opposite a cytosine (8-OxoG/C). The yeast DNA fragment of 1.7 kbp that suppresses spontaneous mutagenesis and overproduces the 8-OxoG/C cleavage activity was sequenced and mapped to chromosome XIII. DNA sequencing identified an open reading frame, designated OGG1, which encodes a protein of 376 amino acids with a molecular mass of 43 kDa. The OGG1 gene was inserted in plasmid pUC19, yielding pYSB110. E. coli (fpg) harboring pYSB110 was used to purify the Ogg1 protein of S. cerevisiae to apparent homogeneity. The Ogg1 protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. The Ogg1 protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Ogg1 protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Ogg1 protein is probably due to the excision of 8-OxoG followed by a beta-elimination at the resulting apurinic/apyrimidinic site.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Genes Fúngicos , N-Glicosil Hidrolasas/biosíntesis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Fúngicos , Clonación Molecular , ADN Glicosilasas , Reparación del ADN , ADN-Formamidopirimidina Glicosilasa , Biblioteca Genómica , Guanina/análogos & derivados , Guanina/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/aislamiento & purificación , Oligodesoxirribonucleótidos , Pirimidinas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Especificidad por SustratoRESUMEN
A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.