LOCATION, DEGREE, AND DIRECTION OF DNA BENDING ASSOCIATED WITH THE HIN RECOMBINATIONAL ENHANCER SEQUENCE AND FIS-ENHANCER COMPLEX

The Fis protein of Escherichia coli and Salmonella typhimurium stimula tes several site-specific DNA recombination reactions, as well as tran scription of a number of genes. Fis binds to a 15-bp core recognition sequence and induces DNA bending. Mutations in Fis which alter its abi lity to bend DNA have been shown to reduce the stimulatory activity of Fis in both site-specific recombination and transcription systems. To examine the role of DNA bending in the activity of the Fis-recombinat ional enhancer complex in Hin-mediated site-specific DNA inversion, we have determined the locations, degrees, and directions of DNA bends a ssociated with the recombinational enhancer and the Fis-enhancer compl ex. Circular-permutation assays demonstrated that a sequence-directed DNA bend is associated with the Fis binding sites in the proximal and distal domains of the recombinational enhancer. Binding of Fis to its core recognition sequence significantly increases the degree of DNA be nding associated with the proximal and distal domains. The degree of D NA bending induced by Fis binding depended on the DNA sequences flanki ng the core Fis binding site, with angles ranging from 42 to 69 degree s. Phasing analyses indicate that both the sequence-directed and the F is-induced DNA bends associated with the proximal and distal domains f ace the minor groove of the DNA helix at the center of the Fis binding site. The positions and directions of DNA bends associated with the F is-recombinational complex support a direct role for Fis-induced DNA b ending in assembly of the active invertasome.