Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.
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.
van der Kemp
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Unité de Recherche Associée 147 Centre National de la Recherche Scientifique, Villejuif, France.
Thomas
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Barbey
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de Oliveira
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Boiteux
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005800:Genes, Fungal; D015698:Genomic Library; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides; D011743:Pyrimidines / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011743:Pyrimidines; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; C037301:N(5)-methyl-N(5)-formyl-2,5,6-triamino-4-hydroxypyrimidine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli