Copley, James and Allinson, Sarah (2018) PIKK-dependent activation of the DNA damage response by long wavelength ultraviolet radiation. Masters thesis, Lancaster University.
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Abstract
Ultraviolet radiation (UV) is one of the main risk factors that increase a person’s chance of developing skin cancer. UV is divided into three subtypes, called UVA, UVB and UVC. UVA and UVB both have the ability to cause different types of DNA damage. Our understanding of UVA and its mechanism of carcinogenesis is poorly understood due to UVB being the focus of research. The DNA damage response (DDR) detects DNA damage, and triggers a signalling pathway which results in cell cycle arrest, DNA repair or apoptosis. The DDR is important for preventing the persistence of UVinduced DNA damage in skin cells. This project used HaCaT cells to investigate UVA and its ability to generate double strand breaks, and how the DDR is activated as a result. Cells exposed to 100 KJ m-2 UVA displayed significant amounts of DSBs, which were largely repaired within 1 hour of irradiation. γH2AX was used as a biomarker of DNA damage, and investigated using Western blotting and immunofluorescence. UVA-induced γH2AX accumulation peaked between 1 and 2 hours, which was seen in the Western blotting. ATM was inhibited using KU-60019, which caused a delay in the accumulation of UVA-induced γH2AX following. UVA-induced CHK2 and p53 activation followed similar patterns in both ATM inhibited and uninhibited samples, and suggested that both components are linked to an ATM independent pathway. UVA-induced PNKP activation was delayed with the addition of an ATM inhibitor, and increased slightly 1 hour post irradiation, suggesting a greater dependence on ATM activity. 53BP1 foci formation was used to investigate NHEJ activation. There was a significant increase in the number of UVA-induced 53BP1 foci, while the addition of an ATM inhibitor prevented this increase, suggesting an ATM dependence for NHEJ activation. This data suggests that UVA generates double strand breaks, which result in the activation of the DDR which peaks within the first 2 hours following UVA irradiation. Furthermore, ATM is essential for the activation of NHEJ, but is not the only kinase responsible for the activation the DDR.