Analyses of Site-Specific Recombination

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DESCRIPTION (provided by applicant): Site-specific recombination systems are important in controlling such diverse processes such as plasmid maintenance, DNA amplification, chromosome segregation, movement of conjugative transposons and integration of genes in integrons. A long-range goal of this research is to understand how bacteriophage Lambda Int performs site-specific recombination. The findings will have a significant impact on other site-specific recombination systems that are members of the diverse Lambda Int family. Biochemical and genetic approaches will be used to characterize the protein-protein and protein-DNA interactions that occur both during the assembly of recombination complexes (intasomes) and the processes of strand cleavage and ligation. Int mutants will be characterized in assays that are designed to determine the defects of individual proteins in the recombination pathway. The host-encoded integration host factor (IHF) also participates in formation of intasomes by inducing bends in the DNA. We will crystallize mutant proteins that have expanded recognition specificities in order to understand how interactions of amino acids within the protein mediate recognition of DNA. Site-specific excision reactions are important for the spread of elements from one genome to another. We will analyze interactions of the excisionase (Xis) protein with INt and DNA. With the exception of phage Lambda (Xis) protein, little is known about how Xis proteins function during the excision reaction. In order to expand our understanding of excision reaction complexes and reactions, we plan to analyze the molecular mechanism of Xis function of page P22 Xis protein. Finally, we will initiate a project on the conjugative transposon CTnDOT. Our preliminary work indicates that although CTnDOT has an integrase that is related to the Lambda Int, it appears to have some mechanistic differences. We plan to develop an in vitro system to study the mechanism of strand exchange and the excision reaction catalyzed by the CTnDOT Int protein.