Characterisation of UVA-induced DNA damage responses in human skin cells
Conmy, Sarah Angela
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Accumulating evidence indicates that exposure to long-wavelength ultraviolet A (UVA) radiation (315-400 nm), which accounts for >95% of solar UV radiation reaching the earth's surface, is a significant risk factor for the development of skin cancer, particularly melanoma. While it is well established that UVA radiation induces DNA damage, including cyclobutane pyrimidine dimers (CPDs) and 8-oxoguanosine (8-oxo-G) lesions, the response of cells to UVA-induced DNA damage is not well characterised on a molecular level. This research characterised UVA radiation-induced DNA damage response (DDR) pathways in human skin cells, including primary human melanocytes. DNA polymerase eta (pol [eta]) is a specialised translesion synthesis polymerase which carries out error-free replication past UVC-induced dithymidine CPDs in vitro and in vivo. The data presented here provides evidence that pol [eta] plays a role in the replication of UVA-damaged DNA in human fibroblasts. Pol [eta] was localised to discrete nuclear foci following UVA exposure, exclusively in cells undergoing DNA replication. UVA exposure induced a rapid and transient decrease in DNA replication. Pol [eta]-deficient cells showed delayed recovery from UVA-induced replication arrest, and enhanced phosphorylation of checkpoint kinase 1 (Chk1). Moreover, pol [eta]-deficient cells were hypersensitive to UVA radiation in the presence of caffeine, an inhibitor of phosphoinositol 3-kinase-like kinase (PIKK)-dependent Chk1 phosphorylation. This indicates that in the absence of pol [eta], the Chk1 pathway plays an important role in the response of cells to UVA radiation. Overall, the data provides evidence that tolerance of UVA-induced DNA damage is mediated, in part, by pol [eta]. This highlights the importance for UVA protection, especially in individuals with the xeroderma pigmentosum variant disease, lacking pol [eta]. Exposure to UVA radiation is a specific risk factor in the development of melanoma, which arises from malignant transformation of melanocytes. However, the molecular mechanisms linking UVA exposure and melanoma development remain unclear. Here the responses of primary normal human epidermal melanocytes (NHEM) to UVA-irradiation were characterised. UVA radiation induced PIKK-mediated DNA damage responses in NHEM, including activation of both the ATR-Chk1 and ATM-Chk2 pathways. Moreover, a key role was identified for Chk1 in the regulation of DNA replication in UVA-irradiated primary human melanocytes. Using a phospho-proteomic screen, UVA-induced alterations in the phosphorylation status of proteins involved in a number of signalling pathways, including CDK-mediated cell cycle regulation, the mitogen activated Raf-MEK-ERK pathway and the cytoskeleton-associated PKC-[alpha]-adducin pathway, were identified. This research provides insights into the cellular pathways that normally modulate the response of primary human melanocytes to UVA radiation.