Intellectual merit. DNA is under constant chemical assault from cellular and environmental DNA damaging agents. To maintain integrity of their genomes, all organisms possess damage-specific DNA repair proteins faced with the common challenge of locating a particular lesion within a vast excess of "normal" DNA. This research addresses this fundamental question through structure-function analysis of a newly discovered enzyme from Bacillus cereus named AlkD; this enzyme, which belongs to a group of proteins referred-to as DNA glycosylases, will recognize certain DNA bases that have been chemically modified. DNA glycosylases initiate a particular repair pathway by detecting and removing the modified bases from DNA. The basis for glycosylase specificity is poorly understood, but is believed to result in part from instability of the damaged bases themselves. X-ray crystal structures of AlkD bound to damaged DNA, determined in the PI's laboratory, revealed that AlkD utilizes a new DNA binding architecture, referred-to as a HEAT repeat, to capture DNA damage by an unprecedented mechanism. HEAT repeat proteins have recently been identified to form expansive domains within several DNA damage response proteins. Thus, AlkD represents a new class of DNA processing enzyme that offers an opportunity to determine 1) the molecular details by which HEAT repeats engage DNA damage, and 2) the chemical and physical determinants of damaged base recognition and removal. A combination of structural biology methods, including X-ray crystallography and NMR, will be used to obtain high-resolution structures of AlkD complexes with damaged DNA, and the structural data will guide biochemical analyses of DNA binding and base excision activities. Results from this research program will be placed in the context of on-going DNA repair research in the PI's laboratory and elsewhere.
Broader impact. Students are typically only exposed to the theoretical aspects of X-ray crystallography and advanced structural techniques in a lecture setting. This project will integrate research and education by providing practical structural biology training to students at all levels in both the laboratory and the classroom. A hands-on X-ray crystallography module will be incorporated into the PI's protein chemistry course to provide undergraduate and graduate students with the unique opportunity to participate directly in all aspects of protein structure determination. Students will aid in crystallization, structure solution, model building, refinement, and validation of protein-DNA complexes as part of the AlkD research program, and will be directed in groups by both the PI and graduate students in the PI's laboratory. Graduate students associated with this award will direct undergraduates working on their own independent projects in the PI's laboratory in fulfillment of research course credit. Thus, in addition to undergraduates gaining practical research experience in X-ray crystallography, graduate students will gain invaluable teaching experience by guiding students in the laboratory. The PI's affiliations with the College of Arts and Science and five research centers in the medical school at Vanderbilt University offer a rich research and teaching environment involving undergraduates, graduate students postdoctoral fellows, and visiting scientists.