Interactive competition between homologous recombination and non-homologous end joining.
DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in double-strand break (DSB) repair by non-homologous end joining (NHEJ). DNA-PKcs defects confer ionizing radiation sensitivity and increase homologous recombination (HR). Increased HR is consistent with passive shunting of DSBs from NHEJ to HR. We therefore predicted that inhibiting the DNA-PKcs kinase would increase HR. A novel DNA-PKcs inhibitor (1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone; designated IC86621) increased ionizing radiation sensitivity but surprisingly decreased spontaneous and DSB-induced HR. Wortmannin also inhibits DNA-PKcs and reduces DSB-induced HR. IC86621 did not affect HR product outcome, indicating that it affects HR initiation. Thus, HR is increased in the absence of DNA-PKcs, but decreased when DNA-PKcs is catalytically inactive, suggesting interactive competition between HR and NHEJ. The effects of IC86621 and wortmannin were proportional to the level of DNA-PKcs, consistent with inhibited DNA-PKcs acting in a dominant negative manner. We propose that inhibition of DNA-PKcs blocks its autophosphorylation, prevents dissociation of DNA-PKcs from DNA ends, and thereby blocks both HR and NHEJ. By blocking the two major DSB repair pathways, DNA-PKcs inhibitors should radiosensitize at all cell-cycle stages and are therefore excellent candidates for augmenting cancer radiotherapy.
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Chris
C
Allen
James
J
Halbrook
Jac A
JA
Nickoloff
D000098:Acetophenones / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009025:Morpholines / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011995:Recombination, Genetic / Q000235:genetics*; D000077191:Wortmannin
C487214:1-(2-hydroxy-4-morpholin-4-ylphenyl)ethanone; D000098:Acetophenones; D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009025:Morpholines; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin