title: A motif in the C-terminal domain of phiC31 integrase controls the directionality of recombination.
abstract: Bacteriophage C31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, C31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of C31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein-protein interactions.
authors.affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB252ZD, UK.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Rowley
authors.forename: Matthew C A
authors.initials: MC
authors.lastname: Smith
authors.forename: Ellen
authors.initials: E
authors.lastname: Younger
authors.forename: Margaret C M
authors.initials: MC
authors.lastname: Smith
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology*; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D013302:Streptomyces / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D019426:Integrases