IN-VIVO IDENTIFICATION OF INTERMEDIATE STAGES OF THE DNA INVERSION REACTION CATALYZED BY THE SALMONELLA HIN RECOMBINASE

The Hin recombinase catalyzes a site-specific recombination reaction t hat results in the reversible inversion of a 1-kbp segment of the Salm onella chromosome. The DNA inversion reaction catalyzed by the Salmone lla Hin recombinase is a dynamic process proceeding through many inter mediate stages, requiring multiple DNA sites and the Fis accessory pro tein. Biochemical analysis of this reaction has identified intermediat e steps in the inversion reaction but has not let revealed the process by which transition from one step to another occurs. Because transiti on from one reaction step to another proceeds through inter-actions be tween specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hi n recombinase that failed to carry out the DNA exchange reaction. We g enerated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to d efine contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a mo del that Fis protein enhances the binding of Hin to the hixR recombina tion site. These studies identified regions within the Hin recombinase involved ill specific transition steps of the reaction and provided n ew insights into the molecular details of the reaction mechanism.