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 coil. A key feature of most models of recombination for the family of recombinases to whic h XerC and XerD belong is that a Holliday junction forms at the positi on 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 e xchanges. We have tested this hypothesis for Xer recombination by stud ying 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 inf er that the Holliday junction need not be at this position during stra nd cleavage and exchange. With substrates in which the Holliday juncti on is constrained to a region away from the XerC-mediated cleavage poi nt, strand exchange generates products with the predicted mispaired ba ses.