Hughes 9603585 The Salmonella Hin recombinase catalyzes a site-specific DNA recombination event resulting in the reversible inversion of a 1-kilobase segment of the chromosome. This reversible chromosomal inversion event results in a regulatory switch known as flagellar phase variation, which is the alternate expression of either one of two antigenically distinct flagellin antigens expressed on the bacterial cell surface. The long-term goal of this project is to understand in molecular detail the mechanism of DNA strand exchange catalyzed by the Hin recombinase during the chromosomal inversion process. The approaches described in this project are novel and involve the development of sophisticated genetic tools that allow the dissection of individual steps of the DNA inversion reaction in vivo. DNA inversion is thought to occur in the following steps: 1) Hin dimerizes and binds to 26 base pair hix recombination sites that flank the invertible segment of the chromosome; 2) Fis (Factor for inversion stimulation) then binds its target DNA sites within a DNA sequence known as the recombinational enhancer; 3) Synapsis occurs: Hin dimers interact to form tetramers bringing hix recombination sites within local proximity; 4) Invertasome formation: Hin bound to hix sites interacts with Fis bound to the DNA enhancer segment to generate the formation of a nucleoprotein intermediate competent for DNA cleavage; 5) DNA cleavage; 6) DNA strand rotation and exchange; 7) DNA religation; 8) Nucleoprotein complex dissolution. In this project genetic tools are described that allow the identification of mutants in Hin and Fis that cannot catalyze one of the steps in the recombination reaction. Using these tools, the step for which a specific hin or fis mutant is defective can be identified directly, without requiring protein purification and biochemical characterization of each mutant. Through standard molecular techniques the specific amino acid defect in Hin and Fis is determined. The characterization of a large number of mutants allows the identification of the amino acids in Hin and Fis required for specific steps in the recombination process. Finally, this collection of mutants will be used in structure/function studies as part of a detailed molecular characterization of each step in the Hin-mediated DNA recombination reaction. This work will provide insight applicable to areas of biology in which DNA strand exchange is a fundamental component of the biological process under investigation.
