This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. DNA rearrangement/recombination enzymes alter covalent structure of DNA, by cleaving and rejoining DNA strands. These proteins play important roles in diverse biological contexts, including viral integration into host's genome, maintenance of chromosome integrity, and generation of genetic diversity. Using x-ray crystallography, we are studying three distinct classes of DNA rearrangement enzymes to better understand how these enzymes function. The first class is the retroviral integrase. Retroviruses, including HIV-1 that causes AIDS, have an RNA genome that is reverse-transcribed into a linear viral DNA upon entering the host cell. Integration of this viral DNA into host's chromosome is an essential step in the lifecycle of retroviruses, and is carried out by the virally encoded integrase (IN) protein. We are pursuing crystal structures of the functional 3-domain IN protein as well as IN-DNA complexes, using IN from HIV and RSV (Rous Sarcoma Virus) systems. The second class is the DNA resolvase involved in the maintenance of bacterial linear chromosomes. Some bacterial pathogens, including the Lyme disease spirochete Borrelia burgdorferi, have linear chromosomes with covalently closed hairpin termini. Replication of such linear chromosomes requires resolution of a catenated circular intermediate into unit-length chromosomes, which is carried out by the hairpin-forming DNA resolvase enzyme. Using Borrelia and Agrobacterium systems, we are determining crystal structures of the resolvase-DNA complexes to gain insights into the mechanism of DNA strand cleavages and subsequent hairpin formation. The third class is the Holliday junction resolvase involved in resolution of the cruciform-shaped DNA recombination intermediates. We are pursuing crystal structure of the poxvirus HJ resolvase in complex with HJ DNA substrates. If successful, it will be the first atomic structure of an enzyme-DNA complex for the large family of HJ resolvases represented by E. coli RuvC.
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