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Title: A study of the distribution of DNA damage, repair and epigenetic changes following exposure to ultraviolet radiation
Authors: Alhegaili, Alaa Saud
Supervisors: Jones, George DD
Award date: 2-Mar-2016
Presented at: University of Leicester
Abstract: The distribution of DNA damage and repair has been reported to occur heterogeneously. Commonly available techniques, such as comet assay, allow for the measurement of DNA damage at the global, whole-genome level; but this does not reflect important events that happen at the gene/sequence-specific level. In this study, an assay based on a combination of DNA Immunoprecipitation plus next-generation sequencing (DIP-Seq) was developed to assess the induction and repair of DNA damage induced by solar-simulated radiation (SSR) at the genome-wide, sequence-specific level. The global induction and repair of DNA damage including cyclobutane thymine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesions following irradiation with UVB and SSR was assessed via an enzyme-modified comet assay. The results showed that significant levels of DNA damage were induced and that these damages were repaired within 48 h post exposure to SSR. Furthermore, ELISA was used to quantify the global level of CPDs following UVR exposure. Additionally DIPqPCR, as a prelude to DIP-Seq was performed to assess the induction of CPD in response to UVB, and the results again showed a good dose response. Then the DIP-Seq was performed to assess CPD induced by SSR in isolated and cellular DNA. CPD repair assessed by DIP-seq showed gene-dependent highly variable removal of CPDs with high resolution. Also, the effect of SSR irradiation on the levels of histone modifications, H3K9 acetylation and H3K4 trimethylation in HaCaT cells, was quantified by chromatin immunoprecipitation (ChIP) followed by qPCR. Western blot analysis was used to confirm the ChIP assay results, and to assess the global level of histone modification in HaCaT cells after UVR irradiation. Overall, the findings of this project revealed a good agreement between both the global and sequence-specific measurements of SSR-induced DNA damage and its repair, and also between global and sequence-specific measurements of the epigenetic modifications assessed.
Type: Thesis
Level: Doctoral
Qualification: PhD
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Cancer Studies & Molecular Medicine
Leicester Theses

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