DNA in cells is continuously being damaged by diverse sources including UV from sun, industrial pollutant, cigarette smoke, and burnt food. These DNA lesions, if left unrepaired, can block important cellular functions and create errors in the genetic code, which may lead to cell death or diseases. This project will tackle two key questions in the field of DNA repair: How do repair enzymes detect a small number of DNA lesions in a huge "genomic ocean" of normal DNA? Why are some lesions repaired very efficiently, whereas others are not repaired at all? Using innovative, multi-disciplinary approaches, the research will transform the current static view of the lesion detection process into a 3-D molecular trajectory "movie" in unprecedented detail, thus providing important insights that might eventually lead to development of better ways to prevent the occurrence of DNA damaged-induced cellular defects. Equally important benefits will come from Integration of the research into creative education and outreach programs to promote cross-disciplinary training in Chemistry and Physics for graduate and undergraduate students and to build educational modules, incorporating crosscutting concepts of the new Next Generation Science Standards, to be used by K-12 teachers and their students in the Chicago Public Schools.
The XPC complex recognizes diverse, environmentally induced DNA lesions from the genomic DNA, and thus is a key to the initiation of the eukaryotic nucleotide excision repair pathway. The recognition efficiency of the lesions can vary widely depending on the lesion, and certain lesions can evade detection by XPC and thus become resistant to nucleotide excision repair. Previous studies from this research group have suggested a novel "kinetic gating" mechanism in which XPC's ability to discriminate between lesions and normal sites may lie in the kinetics of forming an "open" conformation with the damaged (or normal) nucleotides flipped out. The goal of this project is to rigorously investigate this model by a unique combination of complementary technologies including X-ray crystallography, time-resolved temperature-jump fluorescence spectroscopy and chemical crosslinking. Specific aims are (1) to characterize and compare the dynamics of XPC-induced DNA opening in damaged and normal DNA, (2) to determine the dynamics and structures of lesion recognition using mutant XPC that lack key DNA-binding structural elements, and (3) to elucidate and compare the structure and kinetics of XPC's binding to repair-resistant and -proficient lesions. The results will provide new insights into the dynamics of lesion recognition. Moreover, the unique approaches developed in this study will also be relevant to other gene regulatory and maintenance systems, and may help uncover a new paradigm of protein functions controlled by transient interactions, which have escaped previous detection by other methods.
