Site-specific recombination systems are important in controlling such diverse processes such as phage insertion and excision, plasmid maintenance, DNA amplification, chromosome segregation and movement of conjugative transposons. A long-range goal of this research is to understand the mechanism of site-specific recombination reactions. The focus of this proposal is the integrase (DOT Int) encoded by conjugative transposon CTnDOT. DOT Int is a member of the tyrosine family of recombinases but the mechanism of the recombination reaction it performs differs from the one used by most other members of the family. We will study the reaction mechanism of the DOT Int enzyme using a variety of approaches. DNA cleavage and ligation assays will be developed to study the reaction mechanism, to identify the sites of cleavage and ligation and to characterize defects of mutant proteins. Holliday junction intermediates will be assembled using synthetic DNA or accumulated by in vitro reactions using newly discovered peptide inhibitors. Resolution of Holliday junctions in the absence and presence of accessory factors that affect directionality will be studied. The order of strand cleavages in the integration and excision reactions will be determined by analysis of the Holliday junctions made by each reaction. Genetic and in vitro techniques will be used to generate mutant DOT Int proteins. Recombination-defective mutants will be isolated from an in vivo screen and their defects will be determined by biochemical techniques. We will use a homology modeling approach to identify and mutate residues that contact DNA. The Dot Int protein will be purified and crystallized to solve its structure and better to understand the differences between the catalytic region of DOT Int and other tyrosine recombinases. Finally, we will characterize the excision reaction. The Orf2c, Orf2d, and Exc proteins, will be purified and their roles in the excision reaction will be defined. ? Conjugative transposons such as CTnDOT carry various genes that encode resistance to antibiotics and are widespread amongst intestinal bacteria. Because CTnDOT and its relatives transfer themselves to recipient cells by conjugation and recombination they are an important source for the transfer of antibiotic resistance. These studies will increase our understanding of the recombination mechanism and help develop strategies to inhibit the spread of these elements in Nature. ? ? ?
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