Effects of Holliday junction position on Xer-mediated recombination in vitro

Site-specific recombination mediated by XerC and XerD functions in the segregation of circular replicons in Escherichia coli. A key feature of most models of recombination for the family of recombinases to which XerC and XerD belong is that a Holliday junction forms at the position of the first pair of recombinase-mediated strand exchanges and then branch migrates 6-8 bp to the position of the second pair of strand exchanges. We have tested this hypothesis for Xer recombination by studying the effects of junction position on XerC-mediated strand exchange in vitro. Recombination of synthetic Holliday junction substrates in which junction mobility was constrained to a region extending over or removed away from the normal cleavage and exchange point was analysed. All substrates undergo strand cleavage at the normal position. We infer that the Holliday junction need not be at this position during strand cleavage and exchange. With substrates in which the Holliday junction is constrained to a region away from the XerC-mediated cleavage point, strand exchange generates products with the predicted mispaired bases.