Mechanism of interaction between Ku protein and DNA.
The mechanism of interaction between the Ku autoantigenic protein, a heterodimer of noncovalently linked 70,000- and 80,000-dalton subunits, and DNA was studied using immunoaffinity-purified Ku protein and a 300-base pair EcoRI fragment from HeLa cell DNA. In the nitrocellulose filter-binding assay, the Ku protein bound 32P-labeled double-stranded DNA, and much less efficiently single-stranded DNA. The binding of Ku to DNA was dependent on ionic strength and prevented by IgG from patient sera containing anti-Ku antibodies. In competitive assays, using unlabeled nucleic acid competitors, the DNA binding of Ku was not inhibited in the presence of yeast tRNA, synthetic copolymer of poly(A)-poly(dT), or circular plasmid pBR322 DNA, but was inhibited when the plasmid DNA was cleaved with appropriate restriction endonucleases. The inhibitory activities of cleaved plasmid DNA were independent of the configuration or nucleotide sequences at ends but proportional to the number of recognition sites of restriction enzymes used. Footprint analysis demonstrated that Ku protein protected both 3'- and 5'-terminal regions of double-stranded DNA from DNase I digestion. When Ku protein was fractionated electrophoretically, transferred to nitrocellulose filter, and probed with 32P-labeled DNA, only the 70,000-dalton subunit exhibited DNA binding. Thus, the Ku protein appears to recognize selectively ends of double-stranded DNA molecules. Possible functions of the Ku autoantigen in eukaryotic cells are discussed.
D034961:Antigens, Nuclear; D000954:Antigens, Surface / Q000378:metabolism*; D001324:Autoantigens / Q000276:immunology; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004262:DNA Restriction Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G; D007158:Immunologic Techniques; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D009220:Myositis / Q000276:immunology; D009691:Nucleic Acid Denaturation; D009994:Osmolar Concentration; D010957:Plasmids; D029701:Saccharomyces cerevisiae Proteins; D012595:Scleroderma, Systemic / Q000276:immunology; D012965:Sodium Chloride / Q000494:pharmacology; D013577:Syndrome
D034961:Antigens, Nuclear; D000954:Antigens, Surface; D001324:Autoantigens; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; C081647:high affinity DNA-binding factor, S cerevisiae; D012965:Sodium Chloride; D004247:DNA; D004262:DNA Restriction Enzymes; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen