Reduced sulphydryl groups are required for DNA binding of Ku protein.
The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription. The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents. Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide). Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA. Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding. The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting. The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol. These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups. Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo. Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues.
Zhang
W W
WW
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Yaneva
M
M
D034961:Antigens, Nuclear; D001324:Autoantigens; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction
D034961:Antigens, Nuclear; D001324:Autoantigens; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D003545:Cysteine