Characterization and mechanism of action of Drosophila ribosomal protein S3 DNA glycosylase activity for the removal of oxidatively damaged DNA bases.
We recently demonstrated that Drosophila ribosomal protein S3 specifically cleaved duplex oligodeoxynucleotides at sites of 7,8-dihydro-8-oxoguanine (8-oxoGua), presumably due to S3 protein possessing an N-glycosylase activity that is associated with its known apurinic/apyrimidinic (AP) lyase activity. Here we show, using DNA substrates prepared by gamma-irradiation under N2O and analyzed by gas chromatography/isotope-dilution mass spectrometry, that S3 protein efficiently liberates 8-oxoGua as a free base from the damaged DNA substrate. Of the 15 additional modified bases present in the DNA substrate, the only other one acted on by S3 protein was 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Specificity constants measured for the removal of 8-oxoGua and FapyGua indicate that S3 protein has a similar preference for both of these modified purines. Having established that S3 protein contains an N-glycosylase activity, we next examined the repair of duplex oligonucleotides containing 8-oxoGua (8-oxoGua-37-mer) positioned opposite Cyt, Gua, Thy, or Ade. Significant cleavage of the 8-oxoGua-37-mer was only detected for an opposing Cyt. Moreover, we show that an imino covalent enzyme-substrate intermediate is formed between S3 protein and 8-oxoGua-37-mer, a result similar to other DNA repair enzymes that catalyze N-glycosylase/AP lyase-type reactions at sites of DNA damage.
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Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808, USA. deutscwa@mhs.pbrc.edu
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Jaruga
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Zastawny
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Dizdaroglu
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D000818:Animals; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D004330:Drosophila; D008401:Gas Chromatography-Mass Spectrometry; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011743:Pyrimidines / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D011743:Pyrimidines; D012269:Ribosomal Proteins; C044152:ribosomal protein S3; C071023:2,6-diamino-4-hydroxy-5-formamidopyrimidine; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases