DNA topoisomerases are ubiquitous proteins that control and maintain the topological state of DNA in the cell. They are involved in replication, transcription, and genetic recombination. Their role in DNA metabolism has made the important targets of novel chemotherapeutic and antibiotic agents. In Escherichia coli, two type I DNA topoisomerases have been identified and characterized: DNA topoisomerase I and DNA topoisomerase III. Both proteins work by forming a transient phosphotyrosine bond with DNA that allows another strand or duplex to pass through the break. We have identified, characterized, and elucidated the atomic structures of a 67 kDa fragment of E. coli DNA topoisomerase I. The structure suggests an enzyme-bridged mechanism of action for these type of proteins and poses new questions about their detailed catalytic mechanisms and their interactions with DNA. The long term goal of this proposal is to understand the mechanism of action of prokaryotic type I enzymes in atomic detail.
The specific aims of the proposal are: a) To refine the atomic structure of the 67 kDa fragment of E. coli DNA topoisomerase I to 1.8 A; b) To study the interactions between DNA topoisomerase I and DNA; c) To study the chemical and structural determinants of catalytic function by a combination of site-directed mutagenesis, high resolution x-ray analysis, and biochemical and physicochemical studies; d) To solve the structure of E. coli DNA topoisomerase III; and e) To compare the structures of the two enzyme to identify the features important for their similarities and differences. Our work on E. coli DNA topoisomerase I will be largely guided by the recently solve structure of a 67kDa fragment of the enzyme in our laboratory. The crystals of the 67 kDa fragment diffract to 1.8 A and will allow us to obtain a very detailed atomic model on which to base our studies of DNA-protein interactions and the atomic catalytic mechanism. Crystals of the intact E. coli DNA topoisomerase III have already been obtained, and they diffract to at least 3.0 A. The structure determination by x-ray crystallography is in progress.
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