DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination.
DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in repairing double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ). Certain proteins involved in NHEJ are also involved in DSB repair by homologous recombination (HR). To test the effects of DNA-PKcs on DSB-induced HR, we integrated neo direct repeat HR substrates carrying the I-SceI recognition sequence into DNA-PKcs-defective Chinese hamster ovary (V3) cells. The DNA-PKcs defect was complemented with a human DNA-PKcs cDNA. DSB-induced HR frequencies were 1.5- to 3-fold lower with DNA-PKcs complementation. In complemented and uncomplemented strains, all products arose by gene conversion without associated crossover, and average conversion tract lengths were similar. Suppression of DSB-induced HR in complemented cells probably reflects restoration of NHEJ, consistent with competition between HR and NHEJ during DSB repair. Interestingly, spontaneous HR rates were 1.6- to >3.5-fold lower with DNA-PKcs complementation. DNA-PKcs may suppress spontaneous HR through NHEJ of spontaneous DSBs, perhaps at stalled or blocked replication forks. Because replication protein A (RPA) is involved in both replication and HR, and is phosphorylated by DNA-PKcs, it is possible that the suppression of spontaneous HR by DNA-PKcs reflects regulation of replication-dependent HR by DNA-PKcs, perhaps by means of phosphorylation of RPA.
Allen
Chris
C
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Kurimasa
Akihiro
A
Brenneman
Mark A
MA
Chen
David J
DJ
Nickoloff
Jac A
JA
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005785:Gene Conversion / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D009355:Neomycin; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D009355:Neomycin