The more than 500 members of the ? Int Family of recombinases (also referred to as the Tyrosine Family of site-specific recombinases) carry out site-specific recombination in the absence of DNA synthesis or high-energy cofactors and participate in a wide range of biological pathways in prokaryotes, eukaryotes, and archaea. ? Int, which catalyzes integrative and excisive recombination of the ? viral chromosome into and out of the host E. coli chromosome, is one of the well-characterized model systems for a subset of Int Family members that are heterobivalent DNA binding proteins. These recombinases, like ? Int, utilize an ensemble of host- and virally-encoded accessory proteins (IHF, Fis, and Xis in the case of ?) to introduce site-specific bends into their large DNA targets (att sites). The resulting higher-order protein-DNA recombinogenic complexes (approximately 400kDa in size) confer directionality, regulation, and sensitivity to host physiology on the basic Int Family motif of strand exchange. How distal protein and DNA interactions conspire to impose directionality and regulation on the iso-energetic site-specific recombination sponsored by ? Int (and its related siblings) is still not understood. Three different """"""""instruments"""""""" will be used to study the relevant higher-order structures and conformations of recombination complexes. Each one functions over a different distance range: static quenching between a fluorophore and a dark quencher (5-15A);FRET between two fluorophores (20-90A);and cryo-electron microscopy with nano-gold tags (20-1000A). The three specific aims of this proposal are organized around one of these instruments: 1) To construct and compare DNA contour maps of the excisive and integrative recombination complexes at different stages of each reaction;2) To identify the Int-bridged core-arm pairs in recombination complexes at different stages of the excisive and integrative recombination reactions;3) To visualize and compare by cryo-electron microscopy the structural features of the excisive and integrative recombination complexes and their products. In the final analysis all of the data will be pooled to generate informative structures of different intermediates along the reaction pathways of integrative and excisive recombination. There are many members of the ? Int Family that figure prominently in various aspects of health-related issues including the spread of both pathogenicity and antibiotic resistance. This family has also had a very large impact on health-related research from an investigational perspective providing powerful tools for genetic engineering both in vivo and in vitro. The ? Int pathway is a sufficiently well studied model system of this prominent family, to warrant a deep understanding of its underlying molecular mechanisms.
This project aims to understand the detailed workings of the ? site-specific recombination pathway, a model system for a large family of reactions that move genetic material among bacterial species throughout nature. These pathways are responsible for the ubiquitous spread of both pathogenicity and resistance to antibiotics.
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