Protein-DNA Complexes Containing DNA-Dependent Protein Kinase in Crude Extracts from Human and Rodent Cells
| Autor: | Doug W. Chan, Nicholas S. Y. Ting, Joan Allalunis-Turner, Lauri G. Lintott, Susan P. Lees-Miller |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
chemistry.chemical_classification
Radiation Protein subunit Biophysics Biology Ku Protein enzymes and coenzymes (carbohydrates) chemistry.chemical_compound Enzyme chemistry Biochemistry Cell culture Radiology Nuclear Medicine and imaging Radiosensitivity biological phenomena cell phenomena and immunity Protein kinase A DNA Function (biology) |
| Zdroj: | Radiation Research. 151:414 |
| ISSN: | 0033-7587 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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