title: DNA binding and synapsis by the large C-terminal domain of phiC31 integrase.
abstract: The integrase (Int) from phage C31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL x attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein-protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD-CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL x attR. hCTD(E449K) was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality.
authors.affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2BX, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: McEwan
authors.forename: Paul A
authors.initials: PA
authors.lastname: Rowley
authors.forename: Margaret C M
authors.initials: MC
authors.lastname: Smith
mesh_terms: D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic
chemical_list: D004247:DNA; D019426:Integrases