Dissecting the roles of XerC and XerD in Xer site-specific recombination

• Main Author: Ferreira, Henrique
• Other Authors: Sherratt, David J.
• Published: University of Oxford 2002
• Subjects: 572.8; Genetic recombination : Tyrosine : Research
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393446

The tyrosine recombinases XerC and XerD function in the monomerisation of circular dimer replicons in many bacteria. The recombining complex contains two synapsed recombination sites and two molecules each of XerC and XerD. Recombination proceeds through two sequential steps of DNA strand exchanges separated in time and space. A specific pair of recombinases initiates the reaction forming a Holliday junction intermediate, which undergoes a conformational change to allow resolution to recombinant products by the other pair of enzymes. In an attempt to understand the molecular basis of recombination machine assembly and coordination of catalysis, chimeras of XerC and XerD were constructed and their properties studied in partial and complete recombination reactions. XerC and XerD are two-domain proteins, whose C-terminal regions contain all of the catalytic residues. It is demonstrated here that XerC or XerD variants lacking their N-terminal domains are active in recombination when combined with their wild type partners. However, the normal pattern of catalysis is dramatically altered: strand exchange by the recombinase variant is stimulated, while that by the wild type partner is impaired. The primary determinants for the mutant phenotype are shown to reside in the region of a-helix B of XerCD. It is also demonstrated that the exchange of the extreme C-termini of XerCD has a profound effect on the direction of HJ resolution. These observations confirm the importance of the cyclic C-terminal "donor-acceptor" interactions between XerC and XerD. Finally, the recombination reaction catalysed by ResD, a tyrosine recombinase encoded by the F-plasmid of E. coli, which is believed to function in the monomerisation of F-plasmid dimers, was reconstituted in vitro. Recombination is intramolecular and shows topological selectivity. ResD lacks a region corresponding to the N-terminal domains of XerCD, and hence its characterisation might supply further insights about the roles of the N-terminal domains of tyrosine recombinases.