Repair of oxidative DNA damage in Drosophila melanogaster: identification and characterization of dOgg1, a second DNA glycosylase activity for 8-hydroxyguanine and formamidopyrimidines.
In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using gamma-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from gamma-irradiated DNA. with k(ca)(t)/K:(M) values of 21.0 x 10(-5) and 11.2 x 10(-5) (min(-1) nM(-1)), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a ss-elimination reaction at the 3'-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.
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CEA, Département de Radiobiologie et Radiopathologie, UMR217 CNRS-CEA, Radiobiologie Moléculaire et Cellulaire, 60 rue du Général Leclerc, BP6, 92265-Fontenay aux Roses, France.
Dizdaroglu
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Doerflinger
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Boiteux
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Radicella
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JP
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D043382:DNA-Formamidopyrimidine Glycosylase; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000378:metabolism; D010053:Ovary / Q000201:enzymology; D018384:Oxidative Stress; D010641:Phenotype; D011743:Pyrimidines / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D011743:Pyrimidines; C071023:2,6-diamino-4-hydroxy-5-formamidopyrimidine; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli