title: Binding and cleavage of nicked substrates by site-specific recombinases XerC and XerD.
abstract: 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 trap reaction intermediates and show that XerC cleaves the top strand efficiently while XerD is less efficient at cleaving the bottom strand of dif. Recombinase-mediated cleavage positions are separated by six base pairs and occur at either end of the dif central region adjacent to the recombinase binding sites. XerC can cleave the top strand of dif inefficiently 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 two XerC protomers bound to adjacent binding sites, suggesting that a conserved interaction domain is present in both XerC and XerD. Cooperativity between two identical recombinase protomers does not occur on un-nicked linear DNA. Ethylation interference footprinting of two XerD catalytic mutant proteins suggests that the conserved domain II arginine from the integrase family RHRY tetrad may make direct contact with the scissile phosphate.
authors.affiliation: Microbiology Unit, Department of Biochemistry, University of Oxford, UK.
authors.forename: G W
authors.initials: GW
authors.lastname: Blakely
authors.forename: A O
authors.initials: AO
authors.lastname: Davidson
authors.forename: D J
authors.initials: DJ
authors.lastname: Sherratt
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002455:Cell Division; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D019426:Integrases; D009154:Mutation; D045522:Recombinases; D011995:Recombination, Genetic; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D045522:Recombinases; C473360:XerC protein, E coli; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C473361:XerD protein, E coli; C115006:integron integrase IntI1