STRUCTURAL STUDIES OF SITE-SPECIFIC RECOMBINATION

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DESCRIPTION (provided by applicant): Site-specific DNA recombinases related to the phage lambda integrase protein (;-int) have diverse biological functions including the segregation of plasmids and chromosomes, viral integration, regulation of gene expression, and programmed gene rearrangements. These tyrosine recombinases catalyze DNA cleavage and ligation using a tyrosine side chain without the addition of a high energy cofactor. They are the only enzymes known to both create branched Holliday junction intermediates and to resolve them into recombinant DNA products. The DNA recombination activity of ;-int is regulated by interactions with DNA arm sites flanking the site of DNA strand exchange. Accessory DNA bending proteins compact the DNA substrate to enable simultaneous interactions of ;-int with arm-sites and the core-sites of recombination. Arm-site binding interactions make the chemically reversible recombination reaction effectively irreversible. We are developing kinetic assays to test a proposed mechanism for the allosteric control of the ;-int recombinase by DNA binding and bending of the arms. Small angle x-ray scattering methods are being used to map the topology of the arm DNAs wrapping around ; recombination complexes. As a second instructive example, we are studying the related TelK protein, an essential phage replication protein that cleaves dsDNA to generate two hairpin ends. TelK and related telomere resolvases convert concatenated replication intermediates into unit length linear chromosomes with covalently-closed, hairpin ends. Crystal structures of TelK bound to cleaved DNA substrates suggest that a protein-induced distortion of DNA separates the cleaved strands to prevent re-ligation of the dsDNA, enabling formation of hairpin product. Conventional and single molecule assays of DNA binding, cleavage, hairpin folding, and ligation are being developed. Crystal structures will be determined of additional TelK-DNA reaction intermediates. The tyrosine recombinases and TelK catalyze similar chemical reactions, working against the torsional stiffness and thermodynamic stability of the starting double-stranded DNA substrate(s) in order to remodel the DNA. Different outcomes by ;-int and TelK are achieved through different architectures of the enzyme-DNA complexes and their effects on positioning the cleaved strands of DNA. Our combined structure-function analyses of these enzymes will provide a deeper understanding of the energetic coupling between protein-induced distortions of DNA structure and the chemistry of phosphodiester bond cleavage and formation. This knowledge has application in improving site-specific recombination as a gene delivery technology. PUBLIC HEALTH RELEVANCE: We are studying enzymes that cut and rejoin DNA, performing essential tasks in bacteria and viruses. These enzymes have potential genetic engineering applications for inserting genes at precise locations of the human genome. They are also a potential target for the development of new antibiotics that kill bacteria.