Accurate repair of DNA damage is critical to the survival of all living organisms. The proposed research builds upon a successful program that has previously demonstrated the mechanisms by which enzymes reposition and subsequently excise oxidatively damaged bases such as 8-oxoG from duplex DNA. This intricate restorative process has evolved in all cells to preserve the integrity of genomic DNA;in its absence, mutations may accumulate in the cell, contributing to the development of cancer and other chronic diseases. While significant progress has been made in understanding the nature of the DNA repair process, a crucial unsolved problem is the mechanism by which these enzymes specifically recognize their cognate lesions among the vast excess of undamaged DNA. These lesions typically vary from their undamaged analogs by only a few atoms;thus structural biology is a natural choice for unraveling the detailed interactions that provide specificity. However, crystallography has thus far supplied insight primarily into the late stages of lesion recognition, when base eversion or excision already has occurred, and it provides little information about the key energetics that drive the observed interactions. We believe that """"""""recognition"""""""" of damage by DNA glycosylases is not limited to interactions in the enzyme's active site but, rather, is part of a dynamic process involving a series of conformational changes, each of which may ultimately contribute to lesion specificity. To overcome the experimental limitations that have thus far prevented a comprehensive understanding of lesion recognition, we have designed a research plan that complements new experiments with state-of-the-art molecular simulations and energy analysis, which will generate testable hypotheses that we will validate and feed back into the design of new experiments. A key strength of our plan is the integration of recognized experts in each area into a single, coherent research team.
Our specific aims are to characterize structurally and energetically the primary steps in DNA damage recognition, including the initial encounter of enzymes with intrahelical G or 8-oxoG;to characterize structurally, energetically and kinetically the translocation of these enzymes along DNA as they search for lesions;and to similarly characterize the processes by which lesions are everted from duplex DNA, amino acids of the enzymes are inserted into the resulting void in the duplex, and the pre-excision (Michaelis) complex is formed.
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