Site-specific recombination of DNA is an essential event in many biological processes that involve genomic rearrangements. Currently, there is great interest in well-characterized site-specific recombination systems that can be used to target genes to specific locations on eukaryotic chromosomes in vivo. It is therefore important to understand the physical and chemical factors that govern the efficiency, product distribution, and target specificity of recombination. The overall goal of the proposed program is to understand the role of DNA structure in site-specific recombination by the FLP recombinase of S. cerevisiae. FLP is the only eukaryotic site-specific recombinase that has been characterized to any appreciable extent and many aspects of the interaction of FLP with its target site are unknown. This proposal focuses on the role of DNA secondary and tertiary structure in the recombination reaction and on aspects of recombination site sequence that are important for the assembly and organization of functional recombinase-DNA complexes. The structure of the recombinase-DNA synaptic complex (the intermediate nucleo-protein intermediate involved in site pairing and strand exchange) will be studied by a combination of physical and biochemical approaches. Rotational diffusion techniques, which measure the rate of overall rotational motion of macromolecules in solution, will be applied to determine the geometry of DNA in the synaptic complex. This information I will be combined with results from topological and helical phasing studies to obtain a model for the secondary and tertiary structure of DNA in the synaptic complex. The geometry obtained from these studies will be compared to that predicted for four-stranded Holliday intermediates, proposed to be a key intermediate in FLP recombination. Knowledge of the detailed structure of DNA in the synaptic intermediate will be used to explain the effects of certain recombination site mutations that have pronounced and puzzling effects on the rate and efficiency of recombination.
