title: Characterization and mechanism of action of Drosophila ribosomal protein S3 DNA glycosylase activity for the removal of oxidatively damaged DNA bases.
abstract: 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.
authors.affiliation: Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808, USA. deutscwa@mhs.pbrc.edu
authors.forename: W A
authors.initials: WA
authors.lastname: Deutsch
authors.forename: A
authors.initials: A
authors.lastname: Yacoub
authors.forename: P
authors.initials: P
authors.lastname: Jaruga
authors.forename: T H
authors.initials: TH
authors.lastname: Zastawny
authors.forename: M
authors.initials: M
authors.lastname: Dizdaroglu
mesh_terms: 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
chemical_list: 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