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Effects of targeted phosphorylation site mutations in the DNA-PKcs phosphorylation domain on low and high LET
Ian M Cartwright1, Justin J Bell1, Junko Maeda1
1Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA.
Abstract:
The present study investigated the effect of targeted mutations in the DNA-dependent protein kinase catalytic subunit and phosphorylation domains on the survival of cells in response to different qualities of ionizing radiation. Mutated Chinese hamster ovary V3 cells were exposed to 500 MeV/nucleon initial energy and 200 keV/μm monoenergetic Fe ions; 290 MeV/nucleon initial energy and average 50 keV/μm spread-out Bragg peak C ions; 70 MeV/nucleon initial energy and 1 keV/μm monoenergetic protons; and 0.663 MeV initial energy and 0.3 keV/μm Cs137 γ radiation. The results demonstrated that sensitivity to high linear energy transfer radiation is increased when both S2056 and T2609 clusters each contain a point mutation or multiple mutations are present in either cluster, whereas the phosphoinositide 3 kinase cluster only requires a single mutation to induce the sensitized phenotype of V3 cells. Additionally, the present study demonstrated that sensitivity to DNA cross-linking damage by cisplatin only requires a single mutation in one of the three clusters and that additional point mutations do not increase cell sensitivity.
Insights
Targeted mutations in DNA-dependent protein kinase (DNA-PK) affect cell survival. Mutations in specific DNA-PK clusters increase sensitivity to high-energy radiation and DNA damage, impacting cell radiosensitivity.
Area of Science:
- Molecular Biology
- Radiation Biology
- Cell Biology
Background:
- DNA-dependent protein kinase (DNA-PK) is crucial for DNA repair and cell survival.
- Understanding DNA-PK's role in radiation response is vital for cancer therapy.
- Specific phosphorylation sites and domains within DNA-PK are key to its function.
Purpose of the Study:
- To investigate the impact of mutations in DNA-PK catalytic and phosphorylation domains on cell survival.
- To determine how different types of ionizing radiation affect mutated cells.
- To assess the combined effects of radiation and DNA-damaging agents on cell sensitivity.
Main Methods:
- Utilized Chinese hamster ovary V3 cells with targeted mutations in DNA-PK.
- Exposed cells to various ionizing radiation types: Fe ions, C ions, protons, and Cs137 gamma radiation.
- Assessed cell survival and sensitivity to DNA cross-linking agent cisplatin.
Main Results:
- Mutations in S2056 and T2609 clusters increase sensitivity to high linear energy transfer (LET) radiation.
- A single mutation in the phosphoinositide 3-kinase cluster sensitizes V3 cells to radiation.
- Cisplatin-induced DNA cross-linking sensitivity requires only a single mutation in any of the three investigated clusters.
Conclusions:
- DNA-PK mutations differentially affect cellular responses to various radiation qualities.
- The phosphoinositide 3-kinase cluster is particularly sensitive to single-point mutations regarding radiation response.
- Targeted mutations in DNA-PK can significantly alter cell sensitivity to both radiation and chemotherapeutic agents.
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