The effects of symmetrical recombination site hixC on Hin recombinase function

An artificial recombination site hixC composed of two identical half-sites that bind the Hin recombinase served as a better operator in vivo than the wild type site hixL (Hughes, K. T., Youderian, P., and Simon, M. I (1988) Genes & Dev. 2, 937-948). In vitro binding assays such as gel retardation assay and methylation protection assay demonstrated that Hin binds to hixC as tightly as it binds to hixL, even when the sites are located in negatively supercoiled plasmids. However, hixC served as a poor recombination site when it was subjected to the standard inversion assay in vitro. hixC showed a 16-fold slower inversion rate than the wild type. A series of biochemical assays designed to probe different stages of the Hin-mediated inversion reaction, demonstrated that Hin dimers bound to hixC have difficulty in forming paired hix site intermediates. KMnO4 and S1 nuclease assays detected an anomalous structure of the center of hixC only when the site was in negatively supercoiled plasmids. Mutational analysis in the central region of hixC and assays of paired hix site formation with topoisomers of the hixC substrate plasmid suggested that Hin is not able to pair hixC sites because of the presence of the anomalous structure in the center of the site. The structure does not behave like a DNA "cruciform" since Hin dimers still bind efficiently to the site. It is thought to consist of a short denatured "bubble" encompassing 2 base pairs. During the study of mutations in the center of hixC, it was found that Hin is not able to cleave DNA if a guanine residue is one of the two central nucleotides close to the cleavage site. Furthermore, Hin acts in a concerted fashion and cannot cleave any DNA strand if one of the four strands in the inversion intermediate is not cleavable.