ABSTRACT
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 activity are radiosensitive and are defective in DNA double-strand break repair and V(D)J recombination. Although much information regarding the interactions 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 electrophoretic mobility shift assays, that chemical crosslinkers enhance the formation of protein-DNA complexes containing DNA-PKcs, Ku and other proteins in extracts from cells of normal human cell lines. Extracts from cells of the radiosensitive human cell line M059J, which lacks DNA-PKcs, are not competent to form these protein-DNA complexes, while addition of purified DNA-PKcs protein restores complex formation. This assay may be useful for screening for 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 lines due to the low abundance of DNA-PKcs in these cells.
Subject(s)
Antigens, Nuclear , DNA Helicases , DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , 3T3 Cells , Animals , DNA-Activated Protein Kinase , HeLa Cells , Humans , Ku Autoantigen , MiceABSTRACT
The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit of approximately 470 kDa (DNA-PKcs) and the DNA-binding protein, Ku. Absence of DNA-PK activity confers sensitivity to x-rays and defects in both DNA double-strand break repair and V(D)J recombination. However the precise function of DNA-PK in DNA double-strand break repair is not known. Here we show, using electrophoretic mobility shift assays, that polypeptides in a fraction purified from human cells interact with DNA-PK and stabilize the formation of a complex containing DNA-PKcs-Ku and DNA. Five polypeptides in this fraction have been identified by amino-terminal sequence analysis and/or immunoblotting. These proteins are NF90 and NF45, which are the 90- and 45-kDa subunits of a protein known to bind specifically to the antigen receptor response element of the interleukin 2 promoter, and the alpha, beta, and gamma subunits of eukaryotic translation initiation factor eIF-2. We also show that NF90, NF45, and eIF-2 beta are substrates for DNA-PK in vitro. In addition, recombinant NF90 promotes formation of a complex between DNA-PKcs, Ku, and DNA, and antibodies to recombinant NF90 or recombinant NF45 immunoprecipitate DNA-PKcs in vitro. Together, our data suggest 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 DNA-PK.
Subject(s)
Antigens, Nuclear , DNA Helicases , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Autoantigens/metabolism , DNA/metabolism , DNA-Activated Protein Kinase , Eukaryotic Initiation Factor-2/metabolism , Humans , Interleukin-2/genetics , Ku Autoantigen , Macromolecular Substances , NFATC Transcription Factors , Nuclear Factor 45 Protein , Nuclear Factor 90 Proteins , Phosphorylation , Promoter Regions, GeneticABSTRACT
Lack of DNA-dependent protein kinase (DNA-PK) activity confers radiosensitivity and defective DNA double-strand break repair. Nine human malignant glioma cell lines were studied to determine whether differences in DNA-PK activity reflect differences in inherent radiosensitivity or are predictive of tumor treatment response. DNA-PK activity was present in all cell extracts, as were the DNA-PK proteins, DNA-PK catalytic subunit, Ku p70, and Ku p80. No correlation was found between the levels of DNA-PK activity and inherent radiosensitivity or in the tumor treatment response. These preliminary results suggest that variation in DNA-PK activity may not be a determinant of clinical response in malignant glioma.
Subject(s)
DNA-Binding Proteins , Glioma/radiotherapy , Protein Serine-Threonine Kinases/metabolism , Radiation Tolerance , DNA-Activated Protein Kinase , Glioma/enzymology , Humans , Nuclear Proteins , Tumor Cells, CulturedABSTRACT
Replication (or partitioning) temperature-sensitive mutants of pKZ1 were isolated and shown to exhibit incompatibility with the resident plasmid (pSLT) of Salmonella typhimurium LT2. Following displacement of pSLT, the mutant plasmids were effectively eliminated from the cell population by passage at 42 degrees C, yielding plasmid-free isolates.
