RUI: DNA Glycosylase Initiated Repair of Damaged Nucleosomes

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DNA glycosylases act on the front-line in the cellular defense against mutations caused by covalent modification of DNA bases. These enzymes patrol the genome and remove damaged bases by catalyzing cleavage of the N-glycosidic bond linking the damaged base to the deoxyribose ring. DNA glycosylases employ a base-flipping mechanism in which the damaged base is inserted into an extrahelical pocket in the DNA glycosylase active site. This mechanism is complicated by the fact that DNA in eukaryotic cells is not free in solution, but is assembled together with histones and other proteins into periodic arrays of nucleosomes that fold together into a fiber known as chromatin. Each nucleosome consists of 146 base pairs of DNA wrapped tightly around a molecular spool of eight histone proteins. To address the mechanism of how DNA glycosylases repair damaged DNA embedded in chromatin, nucleosomes will be reconstituted in vitro from purified histones and DNA that contains a positioned 8-oxoguanine lesion. The rate of DNA glycosylase catalyzed 8-oxoguanine excision from the nucleosomes will be measured by gel electrophoresis. By varying the position and orientation of the 8-oxoguanine lesion within the nucleosome and constraining the movement of the histones and DNA glycosylase relative to the DNA, it will be possible to test competing models to explain how DNA glycosylases act on damaged nucleosomal DNA.This project addresses a fundamental unanswered question in molecular biology: how do DNA processing enzymes access their substrate when it is assembled in the compact structure of chromatin? While this project is focused on how DNA repair enzymes access damaged DNA, the lessons learned will have implications for how a wide variety of enzymes that need to access DNA are able to find their target DNA sequence in a chromatin environment. This research is being conducted by undergraduates at Harvey Mudd College, a highly selective liberal arts college of science and engineering. Undergraduate researchers will gain experience in biochemistry techniques, creative problem solving, and working together as a team. The equipment, methods, and focus of the project are being incorporated into biochemistry lectures and laboratory courses taught by the principal investigator. Finally, this project supports a campus-wide initiative to promote cross-disciplinary scholarship between chemistry and biology.