XER SITE-SPECIFIC RECOMBINATION IN-VITRO

Two related recombinases, XerC and XerD, belonging to the lambda integ rase family of enzymes, are required for Xer site-specific recombinati on in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a s ynthetic Holliday junction-containing substrate has been developed. Re combination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one correspon ding to the conversion of the Holliday junction intermediate back to t he substrate, has been observed, Recombination reactions using XerC an d XerD derivatives that are mutant in their presumptive catalytic resi dues, or are maltose-binding fusion recombinase derivatives, have demo nstrated that this pair of strand exchanges is catalysed by XerC, The site of XerC-mediated cleavage has been located to between the last nu cleotide of the XerC binding site and the first nucleotide of the cent ral region, Cleavage at this site generates a free 5'-OH and a covalen t complex between XerC and the 3' end of the DNA.