DNA looping by Ku and the DNA-dependent protein kinase.
The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an approximately 460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein-nucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.
Cary
R B
RB
Life Sciences Division, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, NM 87545, USA.
Peterson
S R
SR
Wang
J
J
Bear
D G
DG
Bradbury
E M
EM
Chen
D J
DJ
D034961:Antigens, Nuclear; D004247:DNA / Q000648:ultrastructure*; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D018625:Microscopy, Atomic Force / Q000379:methods; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000378:metabolism / Q000648:ultrastructure*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000648:ultrastructure*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; 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