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Protein-DNA complexes containing DNA-dependent protein kinase in crude extracts from human and rodent cells
N S Ting1, D W Chan, L G Lintott
1Department of Biological Sciences, The University of Calgary, Alberta, Canada.
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.
Insights
DNA-dependent protein kinase (DNA-PK) is crucial for DNA repair. This study develops an assay to detect DNA-PKcs interaction with DNA-bound Ku, aiding in screening for DNA-PK function.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- DNA-dependent protein kinase (DNA-PK) is vital for DNA double-strand break repair and V(D)J recombination.
- DNA-PK comprises a catalytic subunit (DNA-PKcs) and the DNA-binding protein Ku.
- The interaction between DNA-PKcs and DNA-bound Ku remains poorly understood.
Purpose of the Study:
- To investigate the interaction between DNA-PKcs and DNA-bound Ku.
- To develop a functional assay for DNA-PK activity.
- To identify proteins interacting with the DNA-PK-DNA complex.
Main Methods:
- Electrophoretic mobility shift assays (EMSAs) were employed.
- Chemical crosslinkers were used to enhance protein-DNA complex formation.
- Extracts from normal and radiosensitive human cell lines (M059J) were utilized.
Main Results:
- Crosslinkers enhanced the formation of protein-DNA complexes containing DNA-PKcs and Ku in normal human cell extracts.
- Extracts from M059J cells (lacking DNA-PKcs) did not form these complexes.
- Addition of purified DNA-PKcs restored complex formation in M059J cell extracts.
- Human DNA-PKcs can interact with rodent Ku, but the assay is less sensitive in rodent cell lines due to low DNA-PKcs abundance.
Conclusions:
- A novel EMSA-based assay effectively detects DNA-PKcs interaction with DNA-bound Ku.
- This assay can screen for DNA-PK function in human cell lines.
- The assay facilitates identification of proteins interacting with the DNA-PK-DNA complex.
- The findings provide insights into the DNA repair mechanism involving DNA-PK.