title: Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage.
abstract: Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Angela E
authors.initials: AE
authors.lastname: Zolner
authors.forename: Ismail
authors.initials: I
authors.lastname: Abdou
authors.forename: Ruiqiong
authors.initials: R
authors.lastname: Ye
authors.forename: Rajam S
authors.initials: RS
authors.lastname: Mani
authors.forename: Mesfin
authors.initials: M
authors.lastname: Fanta
authors.forename: Yaping
authors.initials: Y
authors.lastname: Yu
authors.forename: Pauline
authors.initials: P
authors.lastname: Douglas
authors.forename: Nasser
authors.initials: N
authors.lastname: Tahbaz
authors.forename: Shujuan
authors.initials: S
authors.lastname: Fang
authors.forename: Tracey
authors.initials: T
authors.lastname: Dobbs
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.forename: Nick
authors.initials: N
authors.lastname: Morrice
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hendzel
authors.forename: Michael
authors.initials: M
authors.lastname: Weinfeld
authors.forename: Susan P
authors.initials: SP
authors.lastname: Lees-Miller
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D012694:Serine / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C101467:XRCC4 protein, human; D012694:Serine; C530380:PNKP protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D045643:DNA Repair Enzymes