Ku entry into DNA inhibits inward DNA transactions in vitro.
Association of the DNA end-binding Ku70/Ku80 heterodimer with the 460-kDa serine/threonine kinase catalytic subunit forms the DNA-dependent protein kinase (DNA-PK) that is required for double-strand break repair by non-homologous recombination in mammalian cells. Recently, we have proposed a model in which the kinase activity is required for translocation of the DNA end-binding subunit Ku along the DNA helix when DNA-PK assembles on DNA ends. Here, we have questioned the consequences of Ku entry into DNA on local DNA processes by using human nuclear cell extracts incubated in the presence of linearized plasmid DNA. As two model processes, we have chosen nucleotide excision repair (NER) of UVC DNA lesions and transcription from viral promoters. We show that although NER efficiency is strongly reduced on linear DNA, it can be fully restored in the presence of DNA-PK inhibitors. Simultaneously, the amount of NER proteins bound to the UVC-damaged linear DNA is increased and the amount of Ku bound to the same DNA molecules is decreased. Similarly, the poor transcription efficiency exhibited by viral promoters on linear DNA is enhanced in the presence of DNA-PK inhibitor concentrations that prevent Ku entry into the DNA substrate molecule. The present results show that DNA-PK catalytic activity can regulate DNA transactions including transcription in the vicinity of double-strand breaks by controlling Ku entry into DNA.
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Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 Route de Narbonne, 31077 Toulouse and the Société Française de Recherches et d'Investissements, Berganton, 33127 Saint Jean d'Illac, France.
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Arzel
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Calsou
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D000730:Androstadienes / Q000494:pharmacology; D034961:Antigens, Nuclear; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair / Q000187:drug effects; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014466:Ultraviolet Rays; D000077191:Wortmannin
D000730:Androstadienes; D034961:Antigens, Nuclear; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D000077191:Wortmannin