The type IB DNA topoisomerase family includes the eukaryotic nuclear and mitochondrial topo I enzymes, the poxvirus topoisomerases, and a new group of poxvirus-like topoisomerases in bacteria. Topo IB enzymes relax DNA supercoils by transiently breaking and rejoining one strand of the DNA duplex via a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. Nuclear topo IB is a validated target for cancer chemotherapy. The poxvirus topo is essential for virus replication and is a promising target for the treatment of smallpox. Among the bacteria that encode type IB topos are Mycobacterium avium and Pseudomonas aeruginosa ,which cause significant disease in humans. This laboratory uses vaccinia virus as a model system to study topo lB. A distinctive feature of the poxvirus enzyme is its stringent specificity in strand cleavage. Vaccinia topo transesterifies at the sequence 5'(C/T)CCTTp. Our long-term goals are to: elucidate the structural basis for DNA transesterification chemistry and CCCTT target site recognition; define the catalytic repertoire of vaccinia topo, especially DNA recombination reactions; dissect the essential role of topo during the vaccinia replicative cycle. Our recent discovery of a type IB topo family in bacteria raises important questions about their structural and evolutionary connection to the poxvirus enzymes, their site-specificity, their biological functions, and their potential as targets for discovery of new antimicrobial drugs.
Five specific aims are proposed herein: (1) Determination of the atomic structure of vaccinia topo bound covalently to its DNA target site. (2) Dissection of the contribution of minor groove contacts, individual phosphates, and individual bases to DNA transesterification and determination the step-size of supercoil release. (3) Analysis of topoisomerase-catalyzed Holliday junction resolution reactions. (4) Phenotypic studies of conditional mutants of vaccinia virus in which the topo gene is under the control of the tetracycline operator/repressor. (5) Biochemical and genetic characterization of the TopIB proteins from Mycobacterium avium, Mycobacterium smegmatis, and Pseudomonas aeurginosa.
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