Environmental exposures and lifestyle choices can result in cellular oxidative stress, characterized by the generation of an abundance of reactive oxygen species (ROS). ROS wreak havoc on the structure of DNA bases, with guanine modification yielding the lesion 8-oxo-7,8- dihydroguanine (8oxoG) being particularly prevalent. If not repaired, 8oxoG is mutagenic, causing G to T transversion mutations that can initiate and promote human disease. Guanine-rich G- quadruplex (G4) forming sequences are enriched at promoter proximal regions of the genome, making these regions hot spots for 8oxoG lesions. The repair of 8oxoG by the base excision repair (BER) pathway on G4 promoter sequences (i.e., VEGF) can modulate transcription, however the molecular level interactions and mechanistic details of the repair activity within the G4 promoter context are not well understood. The overall goal of the proposed research is to characterize the molecular level interactions and coordination events coupling the repair of 8oxoG and gene enhancement at the VEGF promoter in response to oxidative stress. The experiments proposed to address this will be conducted in two phases. During the initial mentored K99 phase, X-ray crystallography, advanced nucleic acid kinetics, single-molecule fluorescence, and a human cell- based transcription assays will be utilized to characterize the activities of APE1 and Pol? on the VEGF G4 promoter (Aim 1). While in the mentored phase, the candidate will also take advantage of the resources available at University of Kansas Medical Center for professional development and will continuously apply these skills through structured teaching, mentoring, data presentation, and writing opportunities. During the non-mentored phase (R00) of the project, technical skills the candidate has gained will be used to elucidate a model for the recruitment of transcription factors to the VEGF G4 promoter sequence (Aim 2). Also, during the R00 phase the candidate will extend these approaches to interrogate how BER is completed on the unique G4 substrate and elucidate the extent of coupling between repair and transcription (Aim 3). These experiments will provide the candidate with the data required for an early independent publication and preliminary data for R-series grants. Importantly, during the R00 phase the candidate will develop independence from their mentor by focusing on the interplay between DNA repair and transcription regulation and shifting their work to study the epigenetic-like role of oxidative DNA lesions as transcription modulators through DNA repair.
DNA damage arising from oxidative stress promotes multiple human diseases, and the cell?s primary defense against these lesions is the base excision repair (BER) pathway. The overarching goal of this study is to elucidate the molecular mechanisms coupling two key biological tasks in response to oxidative DNA damage at certain G-quadruplex promoter sequences ?specifically, BER and the transcription of specific genes. I will employ elegant biochemical and human cell- based approaches to elucidate the crosstalk between DNA repair and transcription, while developing the research and professional skills needed to be a successful independent investigator.
