Protein-DNA complexes containing DNA-dependent protein kinase in crude extracts from human and rodent cells

The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit (DNA-PKcs) and a DNA-binding protein, Ku, Cells lacking DNA-PK acti vity are radiosensitive and are defective in DNA double-strand break repair and V(D)J recombination, Although much information regarding the interacti ons of Ku with DNA ends is available, relatively little is known about the interaction of DNA-PKcs with DNA-bound Ku, Here we show, using electrophore tic mobility shift assays, that chemical crosslinkers enhance the formation of protein-DNA complexes containing DNA-PKcs, Ku and other proteins in ext racts from cells of normal human cell lines. Extracts from cells of the rad iosensitive human cell line MO59J, which lacks DNA-PKcs, are not competent to form these protein-DNA complexes, while addition of purified DNA-PKcs pr otein restores complex formation. This assay may be useful for screening fo r DNA-PK function in cells of human cell lines and for identifying proteins that interact with the DNA-PK-DNA complex. We also show that Ku protein in rodent cells can interact with human DNA-PKcs; however, this assay may be less useful for studying Ku/DNA-PKcs interactions in cells of rodent cell l ines due to the low abundance of DNA-PKcs in these cells. (C) 1999 by Radia tion Research Society.