DNA-DEPENDENT PROTEIN-KINASE INTERACTS WITH ANTIGEN RECEPTOR RESPONSEELEMENT-BINDING PROTEINS NF90 AND NF45

The DNA-dependent protein kinase (DNA-PK) is composed of a large catal ytic subunit of approximately 470 kDa (DNA-PKcs) and the DNA-binding p rotein, Ku. Absence of DNA-PK activity confers sensitivity to x-rays a nd defects in both DNA double-strand break repair and V(D)J recombinat ion. However, the precise function of DNA-PK in DNA double-strand brea k repair is not known. Here me show, using electrophoretic mobility sh ift assays, that polypeptides in a fraction purified from human cells interact with DNA-PK and stabilize the formation of a complex containi ng DNA-PKcs-Ku and DNA. Five polypeptides in this fraction have been i dentified by amino-terminal sequence analysis and/or immunoblotting. T hese proteins are NF90 and NF45, which are the 90- and 45-kDa subunits of a protein known to bind specifically to the antigen receptor respo nse element of the interleukin 2 promoter, and the alpha, beta, and ga mma subunits of eukaryotic translation initiation factor eIF-2. We als o show that NF90, NF45, and eIF-2 beta are substrates for DNA-PK in vi tro. In addition, recombinant NF90 promotes formation of a complex bet ween DNA-PKcs, Ru, and DNA, and antibodies to recombinant NF90 or reco mbinant NF45 immunoprecipitate DNA-PKcs in vitro. Together, our data s uggest that NF90, in complex with NF45, interacts with DNA-PKcs and Ku on DNA and that NF90 and NF45 may be important for the function of DN A-PK.