Pnk1, a DNA kinase/phosphatase required for normal response to DNA damage by gamma-radiation or camptothecin in Schizosaccharomyces pombe.
We report the characterization of Pnk1, a 45-kDa homolog of the human polynucleotide kinase PNKP in Schizosaccharomyces pombe. Recombinant Pnk1 like human PNKP exhibits both 5'-DNA kinase and 3'-DNA phosphatase activities in vitro. Furthermore, we detected 3'-DNA phosphatase activity with a single-stranded substrate in extracts from wild-type yeast, but no activity was detected in pnk1delta strains. We have shown that GFP-tagged Pnk1 like mammalian PNKP localizes to the nucleus. Deletion of pnk1 does not affect cell growth under normal conditions but results in significant hypersensitivity to gamma-radiation or camptothecin, an inhibitor of topoisomerase I, suggesting that Pnk1 plays an important role in the repair of DNA strand breaks produced by these agents. The pnk1 deletion mutants were not hypersensitive to ethyl methanesulfonate, methyl methanesulfonate, or 4-nitroquinoline N-oxide. Expression of human PNKP in pnk1delta cells restores resistance to gamma-radiation or camptothecin, suggesting that the functions of yeast Pnk1 and human PNKP have been conserved.
Meijer
Maria
M
Department of Biochemistry & Molecular Biology and Oncology, University of Calgary Health Sciences Center, Calgary, Alberta, T2N 4N1, Canada.
Karimi-Busheri
Feridoun
F
Huang
Timothy Y
TY
Weinfeld
Michael
M
Young
Dallan
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair / Q000502:physiology*; D004271:DNA, Fungal / Q000187:drug effects / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D029702:Schizosaccharomyces pombe Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004271:DNA, Fungal; D009153:Mutagens; D029702:Schizosaccharomyces pombe Proteins; C448179:PnK1 protein, S pombe; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D002166:Camptothecin