DNA LOOPING BY KU AND THE DNA-DEPENDENT PROTEIN-KINASE

The DNA-dependent protein kinase (DNA-PK) is required for DNA double-s trand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription, The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 he terodimer and an approximate to 460-kDa catalytic subunit (DNA-PKcs), DNA-PK has been hypothesized to assemble at DNA DSBs and play structur al as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capabl e of producing high resolution images of native protein and protein nu cleic acid complexes without staining or metal coating. The AFM provid es a rapid and direct means of probing the protein-nucleic acid intera ctions responsible for DNA repair and genetic regulation, Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-P K in association with DNA. A significant number of DNA molecules forme d loops in the presence of Ku. DNA looping appeared to be sequence-ind ependent and unaffected by the presence of DNA-PKcs, Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku i s capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tetherin g of the broken DNA strands.