BINDING AND CLEAVAGE OF NICKED SUBSTRATES BY SITE-SPECIFIC RECOMBINASES XERC AND XERD

In Xer site-specific recombination two related recombinases, XerC and XerD, catalyse strand cleavage and rejoining reactions at a site, dif, in order to ensure normal chromosome segregation during cell division in Escherichia coli. We have used nicked suicide substrates to tray r eaction intermediates and show that XerC cleaves the top strand effici ently while XerD is less efficient at cleaving the bottom strand of di f. Recombinase-mediated cleavage positions are separated by six base p airs and occur at either end of the dif central region adjacent to the recombinase binding sites. XerC can cleave the top strand of dif inef ficiently in the absence of its partner recombinase during a reaction that does not require intermolecular synapsis. The presence of a nick in the bottom strand of dif allows cooperative interactions between tw o XerC protomers bound to adjacent binding sites, suggesting that a co nserved interaction domain is present in both XerC and XerD. Cooperati vity between two identical recombinase protomers does not occur on un- nicked linear DNA. Ethylation interference footprinting of two XerD ca talytic mutant proteins suggests that the conserved domain II arginine from the integrase family RHRY tetrad may make direct contact with th e scissile phosphate. (C) 1997 Academic Press Limited