Human polynucleotide kinase participates in repair of DNA double-strand breaks by nonhomologous end joining but not homologous recombination.
Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5'-DNA kinase activity and a 3'-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5'-phosphate and 3'-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 "dead-end" complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway.
Karimi-Busheri
Feridoun
F
Department of Experimental Oncology, Cross Cancer Institute, University of Alberta, 11560 University Avenue, Edmonton, Alberta, Canada.
Rasouli-Nia
Aghdass
A
Allalunis-Turner
Joan
J
Weinfeld
Michael
M
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001932:Brain Neoplasms / Q000378:metabolism; D002166:Camptothecin / Q000494:pharmacology; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D005909:Glioblastoma / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000235:genetics* / Q000502:physiology*; D011995:Recombination, Genetic; D012854:Sister Chromatid Exchange
D000972:Antineoplastic Agents, Phytogenic; C468783:H2AX protein, human; D006657:Histones; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D002945:Cisplatin; D002166:Camptothecin