The ( Int Family recombinases carry out site-specific recombination in the absence of DNA synthesis or high-energy cofactors. The more than 100 members of this family (also referred to as the Tyrosine Family of site-specific recombinases) 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. coil 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 300kDa in size) confer directionality, regulation, and sensitivity to host physiology on the basic Int Family motif of strand exchange. This proposal focuses on those features that define the heterobivalent recombinases and distinguish them from other family members, such as Cre, FIp, and XerC/D.
The specific aims are to: 1) Understand the mechanisms of silencing Int function via N-domain inhibition; 2) Investigate Ithe cyclically swapped N-domain structure of the synaptic complex; 3) Define the critical interactions of accessory proteins with each other and with Int; 4) Determine the kinetic and dynamic relationships between successive steps in recombination; 5) Map the DNA path(s) of the recombinogenic complex. The results in Aims 2, 3, and 5 should lead to a global model of the higher-order recombinogenic complex. The results in Aims 1, 2, and 4 should fit this model into the biochemical and biological contexts of directionality, regulation, and modulation of ( site-specific recombination. A clear understanding of this pathway will contribute to our views about the large number of other related DNA transactions, many of which bear on health related issues. ( Int family members figure prominently in the lysogenic phage of pathogenic bacteria, in the spread of antibiotic resistance genes, and the evolution of pathogenic gene clusters.
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