MECHANISMS OF SITE-SPECIFIC RECOMBINATION

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DESCRIPTION (provided by applicant): The Flp ('flip') site-specific recombinase is coded for by the 2 micron circle yeast plasmid. Two key pre- chemical steps in recombination are: (1) recognition of the target DNA site by Flp and (2) establishing a functional dimer interface between Flp neighbors. By directed evolution, it is possible to relax or alter the DNA specificity of Flp and to suppress mutations that weaken intersubunit interactions. We will determine the crystal structures of 'altered specificity' and 'suppressor' Flp variants in association with their cognate DNA substrates. The persistence of the 2 micron circle in yeast is mediated by a partitioning system and an amplification system. Flp recombination is at the heart of the latter. We will apply topological methods to identify potential novel activities of Flp, decatenation (and unknotting), which may further assist in equal plasmid partitioning. Flp recombination is constrained by the need for perfect spacer homology between recombination partners (FRT sites). Normally, the sites assume antiparallel geometry within the planar recombination synapse. One piece of evidence suggests that spacer heterology does not abolish recombination; rather it promotes even rounds of recombination to restore spacer homology and parental configuration. Furthermore, the FRT sites are suggested to have a parallel geometry. We will devise topological tests to resolve this apparent contradiction. All biochemical analyses of Flp have been carried out with naked DNA substrates. The 2 micron plasmid, though, is a nuclear resident, packaged into chromatin. We will study features of recombination carried out in the context of nucleosome assembly and chromatin remodeling. We will complete several mechanistic studies in progress and initiate new studies to shed light on the action and regulation of Flp in vivo in yeast. These investigations will advance our understanding of macromolecular recognition and allostery during site-specific recombination. They will provide new insights for improved application of site-specific recombination as a tool for genetic engineering. Finally, they will set the stage for analyzing recombination in the context of high-order chromatin and native 2 micron circle physiology.