A New Structural Architecture for Recognition of DNA Damage

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This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences.Chemical modification and damage of DNA from a continual onslaught of cellular and environmental agents alters genetic information and threatens all aspects of cellular function. DNA repair proteins exist in all organisms to remove damaged DNA and protect the integrity of the genome. This research aims to understand at the atomic level how DNA repair proteins locate a particular type of DNA damage and initiate the process of fixing it. This program will enhance the educational benefits to society through 1) integration of research and education in the laboratory and the classroom, 2) vertical integration at all levels of training--from undergraduates to postdoctoral associates, 3) practical structural biology experience for undergraduates, 4) community outreach, and 5) participation by women and underrepresented groups, and 6) integration of science and art to enhance scientific communication. The close proximity of the College of Arts and Science and the School of Medicine at Vanderbilt University provides an exceptional collaborative training environment. Practical hands-on X-ray crystallography laboratory modules have been incorporated into the PI's courses to provide students with the unique opportunity to directly participate in all aspects of protein structure determination. All personnel associated with this research are involved in recruiting students from underrepresented groups from regional institutions, and the PI is involved in outreach through interactions with local high, middle, and elementary school students.The long term goal of this research is to determine the mechanisms by which DNA repair enzymes locate and repair aberrant DNA. This research focuses on the structures and functions of a relatively new superfamily of DNA glycosylases, represented by the Bacillus cereus AlkD enzyme, that catalyzes the excision of cationic alkylated DNA nucleobases, including N3-methyladenine (3mA) and N7-methylguanine (7mG), which are among the most prevalent forms of DNA damage. Because of their inherent instability, the basis for recognition and removal of cationic alkylbases from DNA is unknown. The AlkD-related enzymes are unique in that they are the only DNA glycosylases specific for cationic lesions and with the ability to excise bulky modifications. Additionally, these enzymes are constructed from a tandem helical repeat architecture that has emerged as an important nucleic acid processing platform in chromatin remodeling and DNA damage response proteins. The PI's group has established that unlike other DNA glycosylases, AlkD does not need to flip the nucleobase target out of the DNA duplex prior to catalysis. Thus, the AlkD superfamily is an ideal system to study the fundamental requirements for base excision repair of alkylation damage repair, with strong potential to reveal novel mechanistic insight into DNA damage recognition. Four specific aims will integrate structural and computational biology, biochemistry, and genetic approaches to 1) determine the physicochemical features that underlie recognition and excision of cationic N3- and N7-alkylpurines, 2) elucidate the diversity among the AlkC/D superfamily that defines substrate specificity, 3) investigate how this unique damage recognition platform can excise bulky DNA adducts, and 4) understand the apparent redundancy between alkylpurine DNA glycosylase activities in Bacillus and to determine if AlkD participates in alternative repair pathways.