Despite the packaging of eukaryotic DNA into chromatin through repeating units known as the nucleosomes, it is constantly damaged via reactive oxygen species (ROS). 8oxo-guanine (8oxoG) is a common form of DNA damage resulting from the oxidation of guanine. If not repaired, 8oxoG is mutagenic, causing G to T transversion mutations that can initiate and promote genomic instability and ultimately human disease, such as cancer. The cells primary defense against 8oxoG is the base excision repair (BER) pathway. Two BER proteins involved in the initial recognition and removal of 8oxoG are 8oxoG DNA glycosylase 1 (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1). OGG1 and APE1 must find, access, and repair genomic DNA damage in complex chromatin structures, where the DNA is packaged into nucleosomes. Nucleosomes present a significant barrier to the activities of OGG1 and APE1, which is alleviated when the damage is positioned near the nucleosome entry/exit site. Importantly, the entry/exit site is known to be highly dynamic and undergoes spontaneous and reversible unwrapping and rewrapping of the nucleosomal DNA, thus providing access to the DNA for protein binding. Nucleosome dynamics are further regulated through post-translational modifications (PTMs) to the nucleosome, which allow the cell to fine-tune access to the DNA under different cellular conditions. Despite it being critical to understanding how oxidative DNA damage is repaired within chromatin, mechanistic insight into how OGG1 and APE1 accomplish this remains elusive. To this end, the overarching goal of this proposal is to reveal how OGG1 and APE1 access and process DNA damage in a chromatin environment. The proposal is based on the hypothesize that nucleosomal DNA dynamics and histone PTMs are key regulatory determinants for OGG1 and APE1 to access and process DNA damage. To test this hypothesis, three specific aims have been developed that integrate powerful and complementary biophysical techniques to provide extensive insight into DNA damage and repair in chromatin by OGG1 and APE1.
Aim 1 will determine how nucleosomal DNA dynamics regulate OGG1 and APE1 access to DNA damage using single-molecule fluorescence microscopy.
Aim 2 will determine how histone PTMs further regulate DNA damage access and processing by OGG1 and APE1 using single-molecule fluorescence microscopy and DNA enzymology. Finally, Aim 3 will elucidate the molecular basis for OGG1 and APE1 interactions with damaged nucleosomes using cryo-electron microscopy. Completion of these aims will provide a comprehensive understanding of how DNA damage is repaired in the context of chromatin, while providing training in state-of-the-art biophysical techniques. This innovative proposal will be carried out at the University of Kansas Medical Center under the guidance of an excellent mentorship team. In addition to the research component, the proposal incorporates a training plan that emphasizes career and professional development. Ultimately, this proposal will provide the skills and expertise necessary for the applicant to build a productive independent research group at the interface of DNA damage repair and chromatin.
The base excision repair (BER) pathway is the cells primary defense against mutagenic oxidative DNA damage, which has been linked to numerous human disease states, such as cancer. The overarching goal of this proposal is to understand how BER proteins find and process oxidative DNA damage within chromatin, where nucleosomes act as a barrier to the successful repair of oxidative DNA damage by BER proteins. A combination of powerful and complementary biophysical techniques will be utilized to provide molecular insight into how DNA damage is accessed and repaired within chromatin.
