Purification and partial characterization of a DNA 3'-phosphatase from Schizosaccharomyces pombe.
Cells that depend on oxygen for survival constantly produce reactive oxygen species that attack DNA to produce a variety of lesions, including single-strand breaks with 3'-blocking groups such as 3'-phosphate and 3'-phosphoglycolate. These 3'-blocking ends prevent the activity of DNA polymerase and are generally removed by DNA repair proteins with 3'-diesterase activity. We report here the purification and partial characterization of a 45 kDa protein from Schizosaccharomyces pombe total extract based on the ability of this protein to process bleomycin- or H(2)O(2)-damaged DNA in vitro to allow DNA repair synthesis by DNA polymerase I. Further analysis revealed that the 45 kDa protein removes 3'-phosphate ends created by the Escherichia coli fpg AP lyase following the incision of AP site but is unable to process the 3'-alpha,beta unsaturated aldehyde generated by E. coli endonuclease III. The protein cannot cleave DNA bearing AP sites, suggesting that it is not an AP endonuclease or AP lyase. We conclude that the 45 kDa protein purified from S. pombe is a DNA 3'-phosphatase.
Jilani
Arshad
A
Hôpital Maisonneuve-Rosemont, Centre de Recherche, Université de Montréal, 5415 Boul. de l'Assomption, Montreal, Québec H1T 2M4 Canada.
Ramotar
Dindial
D
D002413:Cations, Divalent / Q000494:pharmacology; D003429:Cross Reactions; D004249:DNA Damage; D004260:DNA Repair; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D007106:Immune Sera / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009708:Nucleotidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000276:immunology / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D002413:Cations, Divalent; D007106:Immune Sera; D010710:Phosphates; D029702:Schizosaccharomyces pombe Proteins; D006861:Hydrogen Peroxide; C448179:PnK1 protein, S pombe; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D009708:Nucleotidases; C039170:deoxynucleotide 3'-phosphatase; D008274:Magnesium