Analysis of the mechanism of interaction of simian Ku protein with DNA.
Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.
Paillard
S
S
Institut Jacques Monod, Paris, France.
Strauss
F
F
D000818:Animals; D034961:Antigens, Nuclear; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004270:DNA, Circular / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012492:Salts; D012689:Sequence Homology, Nucleic Acid
D034961:Antigens, Nuclear; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012492:Salts; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen