title: Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining.
abstract: The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.
authors.affiliation: Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Chappell
authors.forename: Les A
authors.initials: LA
authors.lastname: Hanakahi
authors.forename: Feridoun
authors.initials: F
authors.lastname: Karimi-Busheri
authors.forename: Michael
authors.initials: M
authors.lastname: Weinfeld
authors.forename: Stephen C
authors.initials: SC
authors.lastname: West
mesh_terms: D034961:Antigens, Nuclear; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D006801:Humans; D000072200:Ku Autoantigen; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen