If DNA could adopt only the structure of DNA with which we are most familiar -- the canonical Bform double helix -- it would be devoid of biologic function. The ability of proteins to coerce DNA into a variety of non-canonical structures is an absolute prerequisite for many of the most important processing events that take place on the genome, including replication, transcription initiation, DNA repair, recombination, and packaging into chromatin. The long-term goals of this project are to gain a molecular-level understanding of protein/DNA interactions, with a particular focus on the role of DNA distortion in genome function. The proposed studies will make extensive use of disulfide crosslinking technology, which allows the trapping of unstable or ordinarily transient states of protein/DNA interactions. The ability to trap these complexes enables determination of their structures at high resolution. The specific systems chosen for study are: Topoisomerase Ih Type II topoisomerases (Topo II's) serve important roles in maintaining the superhelical state of the genome and in decatenating chromosomes during cell division. Topo II's are the targets of some of the most important drugs used to treat cancer and bacterial infections. The proposed studies will focus on understanding the mechanisms of these enzymes and the drugs that target them. DNA glycosylases. These enzymes initiate the repair of mutagenic base lesions residues in DNA. How the enzymes distinguish their cognate lesions from the vast excess of normal DNA is a subject of the proposed investigation. E. coil Ada. Ada is a DNA repair protein and transcription factor that controls the cellular resistance pathway to mutagenic and toxic methylating agents. The proposed studies aim to understand the molecular basis for Ada action. RAG recombinase. This enzyme catalyzes a spectacular rearrangement of backbone connectivity in DNA, which is essential for the generation of immunologic diversity. The proposed studies aim to illuminate the recombinase reaction mechanism in detail.
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