Alaa S. Alhegaili
INDIGO (University of Illinois at Chicago) ยท 2016
Abstract
The distribution of DNA damage and repair has been reported to occur \nheterogeneously. Commonly available techniques, such as comet assay, allow for the \nmeasurement of DNA damage at the global, whole-genome level; but this does not \nreflect important events that happen at the gene/sequence-specific level. In this study, \nan assay based on a combination of DNA Immunoprecipitation plus next-generation \nsequencing (DIP-Seq) was developed to assess the induction and repair of DNA damage \ninduced by solar-simulated radiation (SSR) at the genome-wide, sequence-specific \nlevel. \nThe global induction and repair of DNA damage including cyclobutane thymine dimers \n(CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesions following \nirradiation with UVB and SSR was assessed via an enzyme-modified comet assay. The \nresults showed that significant levels of DNA damage were induced and that these \ndamages were repaired within 48 h post exposure to SSR. Furthermore, ELISA was \nused to quantify the global level of CPDs following UVR exposure. Additionally DIPqPCR, \nas a prelude to DIP-Seq was performed to assess the induction of CPD in \nresponse to UVB, and the results again showed a good dose response. Then the DIP-Seq \nwas performed to assess CPD induced by SSR in isolated and cellular DNA. CPD repair \nassessed by DIP-seq showed gene-dependent highly variable removal of CPDs with \nhigh resolution. Also, the effect of SSR irradiation on the levels of histone \nmodifications, H3K9 acetylation and H3K4 trimethylation in HaCaT cells, was \nquantified by chromatin immunoprecipitation (ChIP) followed by qPCR. Western blot \nanalysis was used to confirm the ChIP assay results, and to assess the global level of \nhistone modification in HaCaT cells after UVR irradiation. \nOverall, the findings of this project revealed a good agreement between both the global \nand sequence-specific measurements of SSR-induced DNA damage and its repair, and \nalso between global and sequence-specific measurements of the epigenetic \nmodifications assessed.