Structure/Function Studies of Type 1 Topoisomerases

DESCRIPTION (provided by applicant): A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. DNA topoisomerases are such enzymes. They alter DNA topology by catalyzing the cleavage of single-stranded or double-stranded DNA, the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. Due to their central involvement in many crucial cellular processes, they have become the target for the development of novel anti-bacterial and chemotherapeutic agents. The knowledge of the structure and function of topoisomerases promises, not only to further our understanding of proteins that interact with DNA and alter its topological properties, but also to provide important information to aid in the design of new therapeutic agents.This proposal is concerned with the structure and function of type IA DNA topoisomerases. Type IA topoisomerases are ubiquitous proteins found in all organisms, from bacteria to humans. A significant sequence similarity among all type IA topoisomerases suggests a conserved mechanism of action for all of them. Two type IA topoisomerases have been identified in E. coli, DNA topoisomerase I and Ill. We have solved the structure of both enzymes, of complexes with DNA and nucleotides, and of putative intermediates. Based on these structures, we proposed a detailed mechanism that was recently validated by biochemical experiments. Our findings suggest important parallelisms with the mechanism of type It enzymes. We plan to continue our studies of type IA topoisomerases. The specific aims for this proposal are: i) to solve the structure of E. coli DNA topoisomerase I in complex with DNA, ii) to study the interactions of E. coil DNA topoisomerases lit with DNA, iii) to identify and characterize the structural and chemical determinants of catalytic activity and to elucidate their specific role in the topoisomerization reaction, and iv) to identify, characterize, crystallize, and solve the structure of type IA DNA topoisomerases in different stages of the catalytic cycle. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the complexes that we require for our work, and X-ray crvstallography to solve their structures to high resolution.