Mechanism of inhibition of RNA polymerase I transcription by DNA-dependent protein kinase.
DNA-dependent protein kinase represses RNA polymerase I (Pol I) transcription in vitro. To investigate the mechanism underlying transcriptional repression, we compared Pol I transcription in extracts from cells that either contain or lack the catalytic subunit of DNA-PK (DNA-PKcs). ATP-dependent repression of Pol I transcription was observed in extracts from DNA-PKcs-containing but not -deficient cells, required templates with free DNA ends, and was overcome by exogenous SL1, the factor that nucleates initiation complex formation. Order-of-addition experiments demonstrate that DNA-PKcs does not inactivate component(s) of the Poll transcription machinery. Instead, phosphorylated Ku protein competes with SL1 for binding to the rDNA promoter and, as a consequence, prevents initiation complex formation. The results reveal a novel mechanism of transcriptional regulation by DNA-PK. Once targeted to DNA, autophosphorylated Ku may displace positive- or negative-acting factors from their target sites, thereby repressing or activating transcription in a gene-specific manner.
Michaelidis
Theologos M
TM
Division of Molecular Biology of the Cell II, German Cancer Research Center, D-69120 Heidelberg, Germany.
Grummt
Ingrid
I
D034961:Antigens, Nuclear / Q000378:metabolism; D015153:Blotting, Western; D004247:DNA / Q000502:physiology*; D004265:DNA Helicases; D004275:DNA, Ribosomal / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000072200:Ku Autoantigen; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D035463:Pol1 Transcription Initiation Complex Proteins / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011494:Protein Kinases / Q000494:pharmacology*; D012318:RNA Polymerase I / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D035463:Pol1 Transcription Initiation Complex Proteins; C063660:transcription initiation factor TIF-IB; D004247:DNA; D011494:Protein Kinases; D012318:RNA Polymerase I; D010744:Phosphoric Monoester Hydrolases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen