The Hin Invertasome: Protein-Mediated Joining of Distant Recombination Sites at the Enhancer

Abstract

The Hin protein binds to two cis-acting recombination sites and catalyzes a site-specific DNA inversion reaction that regulates the expression of flagellin genes in Salmonella. In addition to the Hin protein and the two recombination sites that flank the invertible segment, a third cis-acting recombinational enhancer sequence and the Fis protein, which binds to two sites within the enhancer, are required for efficient recombination. Intermediates of this reaction were trapped during DNA strand cleavage and analyzed by gel electrophoresis and electron microscopy in order to determine their structure and composition. The analyses demonstrate that the recombination sites are assembled at the enhancer into a complex nucleo-protein structure (termed the invertasome) with the looping of the three segments of intervening DNA. Antibody studies indicated that Fis physically interacts with Hin and that both proteins are intimately associated with the invertasome. In order to achieve this protein-protein interaction and assemble the invertasome, the substrate DNA must be supercoiled.