Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis

Abstract

A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649-1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially The Hin recombinase of Salmonella spp., in conjunction with the Fis and HU accessory proteins, catalyzes a reversible sitespecific recombination reaction that results in the alternate expression of flagellin antigens, a process known as flagellar phase variation. The Hin-Fis site-specific recombination reaction provides a model system for the investigation of the molecular mechanism of genetic recombination. The recombination reaction catalyzed by the Hin recombinase takes place between two chromosomal sites, hixL on the left and hixR on the right, that flank an invertible DNA segment (27). The underlying molecular mechanism of recombination has been shown to require three proteins, Hin, Fis, and HU (14), and three DNA sites, hixL, hixR, and a recombinational enhancer element (RE) (8, 13). Hin dimers bound to the hixL and hixR recombination sites are thought to act with Fis dimers bound to two sites within the RE during the inversion reaction. The role of HU is to bend DNA, facilitating the assembly of a complex between Hin and Fis. Immunoelectron microscopy has permitted visualization of the nucleoprotein intermediate, or invertasome, that is responsible for carrying out strand exchange (10). By analogy to the closely related Gin recombinase system described below, Hin-Fis-directed invertasome formation is thought to trap DNA supercoils such that after recombination four negative supercoils are lost, effectively driving the reaction to completion. The isolated invertasome structure included Hin and Fis, which were colocalized in the crosslinked complex (10). Fis aids the binding of Hin to the hixR site in vivo (23) and activates the Hin dimers within the synaptic complex in order to initiate concerted DNA cleavage (7, 21).In the closely related Gin invertase system, two negative nodes are trapped as a result of invertasome formation (16-18). Strand exchange is initiated after cleavage of the DNA by the recombinase within the invertasome, and single righthanded (clockwise) rotation about the helix axis, followed by religation, results in a net loss of four negative s...

Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

“…The region of Fis responsible for triggering the change in Hin is located at its N-terminus (Osuna et al 1991) and is composed of two antiparallel b-sheets and a loop (Safo et al 1997). It has been suggested recently that Fis contacts Hin at the dimer interface (Nanassy and Hughes 2001).…”

Stepwise Dissection of the Hin-catalyzed Recombination Reaction from Synapsis to Resolution

“…Other substitutions within the dimer interface have been found to provoke uncoordinated cleavage between synapsed hix sites on plasmids, and most inhibit catalysis altogether. 31,37,38,40,41 DNA exchange, ligation, and synaptic complex resolution…”