Collections > Electronic Theses and Dissertations > Biological Responses to Ultraviolet Radiation and the Effect of Intra-S Checkpoint Inhibition on Ultraviolet Radiation-Induced Mutagenesis
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Sunlight emits a spectrum of electromagnetic radiation that includes ultraviolet, visible, and infrared wavelengths. It is widely accepted that the ultraviolet radiation (UVR) component is responsible for the mutagenic and carcinogenic effects of sunlight. UVR is further sub-divided into three wavelength ranges: UVC (100-280 nm), UVB (280-315 nm) and UVA (315-400 nm) and each of these induce differing types and proportions of DNA damage. Despite much research, there is debate about the relative contributions from different wavelengths in initiating and promoting mutagenesis and carcinogenesis. DNA damage responses have been widely characterized using UVC, which is effectively filtered out by the Earth's atmosphere, while comparatively fewer studies have been conducted utilizing UVA and UVB. The research reported here assessed the biological outcomes of irradiating human dermal fibroblasts with UVR of different wavelengths, using DNA damage dosimetry to allow comparison between different irradiation sources. Cylobutane pyrimidine dimer (CPD) and 6-4 pyrimidine-pyrimidone photoproduct (6-4PP) densities were quantified and used as biomarkers following irradiation with lamps emitting UVA, UVB, or UVC. Comparing fluences that produced equal CPD densities, cytotoxicity, intra-S phase checkpoint response, and mutagenesis were assessed. Regardless of the source of UVR, results showed a striking similarity in the biological outcomes measured when exposures were compared on the basis of CPD dosimetry, suggesting that this UV-induced photolesion significantly contributed to the measured effect. The S-phase checkpoint response to UVR is a signaling cascade that recognizes damaged DNA or abnormal DNA structures and halts or slows new/ongoing DNA replication, potentially reducing the chance that damaged DNA will be replicated prior to its repair. Experiments reported here tested the hypothesis that this checkpoint is a protective system that reduces UVR-induced mutagenesis. Essential S-phase checkpoint components ATR or CHK1 were depleted using siRNA, or the CHK1 kinase pharmacologically inhibited using TCS2312, thereby inhibiting the S-phase checkpoint response to UVR. Subsequently, the UVR-induced mutation frequency at the HPRT locus was measured. Results showed that despite reversing the S-phase checkpoint response to UVR, the mutation frequency in the irradiated population was not increased, challenging the broadly accepted role of this signaling cascade in minimizing mutation burden.