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.