C-terminal interactions between the XerC and XerD site-specific recombinases.
Studies of the site-specific recombinase Cre suggest a key role for interactions between the C-terminus of the protein and a region located about 30 residues from the C-terminus in linking in a cyclical manner the four recombinase monomers present in a recombination complex, and in controlling the catalytic activity of each monomer. By extrapolating the Cre DNA recombinase structure to the related site-specific recombinases XerC and XerD, it is predicted that the extreme C-termini of XerC and XerD interact with alpha-helix M in XerD and the equivalent region of XerC respectively. Consequently, XerC and XerD recombinases deleted for C-terminal residues, and mutated XerD proteins containing single amino acid substitutions in alphaM or in the C-terminal residues were analysed. Deletion of C-terminal residues of XerD has no measurable effect on co-operative interactions with XerC in DNA-binding assays to the recombination site dif, whereas deletion of 5 or 10 residues of XerC reduces co-operativity with XerD some 20-fold. Co-operative interactions between pairs of truncated proteins during dif DNA binding are reduced 20- to 30-fold. All of the XerD mutants, except one, were catalytically proficient in vitro; nevertheless, many failed to mediate a recombination reaction on supercoiled plasmid in vivo or in vitro, implying that the ability to form a productive recombination complex and/or mediate a controlled recombination reaction is impaired.
Spiers
A J
AJ
Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Sherratt
D J
DJ
D000595:Amino Acid Sequence; D002384:Catalysis; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005798:Genes, Bacterial; D019426:Integrases; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D045522:Recombinases; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; 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