Induction and repair of clustered DNA damage sites after exposure to ionizing radiation

• The mechanisms that maintain genomic stability safeguard cells from constant DNA damage produced by endogenous and external stressors. Therefore, this thesis aimed to specifically address questions regarding the requirement and involvement of DNA repair proteins in the repair of various types of radiation-induced DNA damage.The first aim was to determine whether the phosphorylation of DNA-PKcs, a major kinase involved in non-homologous end joining pathway, can be utilized to score the DNA double-strand break (DSB) content in cells. DNA-PKcs phosphorylated (pDNA-PKcs) at T2609 was more sensitive to the cellular DSB content than ɣH2AX, as analyzed by flow cytometry. Further, pDNA-PKcs at T2609 could discriminate between DSB repair-compromised and normal cells, confirming that the pDNA-PKcs can be used as a DSB repair marker. In paper II, the DSB repair was assessed in cells with reduced levels of DNA-PKcs. The reduction in DNA-PKcs resulted in decreased cell survival and unaffected DSB repair. These results clearly indicate that DNA-PKcs plays an additional role in promoting cell survival in addition to its function in DSB repair.The second part of the thesis focused on the characterization of complex DNA damage. DNA damage was investigated after exposure to α-particles originating from Ra-223. The Ra-223 treatment induced a nonrandom DSB distribution consistent with damage induced by high-linear energy transfer radiation. The exposure to Ra-223 significantly reduced cell survival in monolayers and 3D cell structures. The last paper unraveled the fate of heat-sensitive clustered DNA damage site (HSCS) repair in cells. HSCS repair was independent of DSB repair, and these lesions did not contribute to the generation of additional DSBs during repair. Prolonged heating of DNA at relatively low temperatures induced structural changes in the DNA that contributed to the production of DNA artifacts.In conclusion, these results demonstrate that DNA-PKcs can be used to monitor DSB repair in cells after exposure to ionizing radiation. However, the functions of DNA-PKcs are not limited to DSB repair, as it can promote cell survival through other mechanisms. The complexity of the DNA damage produced by high-LET radiation is a major contributor to cell death. However, not all clusters produced in irradiated cells are converted into DSBs during repair.

Nyckelord

NHEJ

DSB repair

clustered DNA damage

DNA repair

DNA-PKcs

HSCS

Ra-223

ionizing radiation

Medicinsk vetenskap

Medical Science

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