DNA-PKcs-OBA/Ku associate in the absence of DNA, as revealed by two-dimensional capillary gel electromobility shift assay.
Ors-binding activity (OBA) has been previously purified by its ability to specifically interact with A3/4, a 36-bp mammalian origin consensus sequence [1]. Peptide sequence analyses identified OBA as Ku86, the largest subunit of Ku antigen, a heterodimeric protein (Ku70/Ku86) involved in several autoimmune disorders [2-5]. The affinity-purified fraction containing OBA/Ku is also enriched for DNA-dependent protein kinase DNA-PKcs, the catalytic subunit of the DNA-PK holoenzyme, of which Ku antigen is the DNA-binding subunit [6-8]. Glycerol-gradient sedimentation analyses have demonstrated the presence of OBA/Ku in a high-molecular-weight complex. In order to investigate whether OBA/Ku and DNA-PKcs are associated in this fraction, we have used a modification of the two-dimensional gel electrophoresis technique originally described [9]. Electromobility shift assays were developed in native capillary gels, which were subsequently used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. The gels were then processed for Western blotting using the Ku70, Ku86 and DNA-PKcs antibodies. This approach has revealed the association of OBA/Ku and DNA-PKcs to give rise to the DNA-PK holoenzyme irrespective of the presence, or the absence of DNA. Altogether, we have proven the utility of this technique for the study of protein-protein and protein-DNA interactions.
Ruiz
Marcia T
MT
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Nichols
Amanda
A
Price
Gerald B
GB
Zannis-Hadjopoulos
Maria
M
D034961:Antigens, Nuclear / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D008970:Molecular Weight; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D021122:Protein Subunits
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D021122:Protein Subunits; 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