A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination

Nucleic Acids Research, Jul 2008

Bacteriophage ϕC31 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, ϕC31 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 ϕC31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein–protein interactions.

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A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination

Nucleic Acids Research A motif in the C-terminal domain of rC31 integrase controls the directionality of recombination Paul A. Rowley 0 Matthew C. A. Smith 1 Ellen Younger 0 Margaret C. M. Smith 0 0 Institute of Medical Sciences, University of Aberdeen , Foresterhill, Aberdeen AB252ZD 1 Institute of Genetics, University of Nottingham , Nottingham NG7 2UH , UK Bacteriophage rC31 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, rC31 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 rC31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein-protein interactions. INTRODUCTION In order to establish a lysogenic life style, many bacteriophages encode an integrase to integrate the phage genome into the host chromosome ( 1,2 ). In association with an accessory factor, the recombination directionality factor (RDF) or Xis, the integrase also excises the phage genome on induction into the lytic pathway ( 2,3 ). During integration, the phage integrases act site-specifically by recombining the host and phage attachment sites, attB and attP, to form the hybrid products attL and attR. In excision attL and attR recombine to regenerate attB and attP. Phage integration and excision has to be a highly regulated process to ensure efficient switching between the lytic pathway and lysogeny ( 1 ). As some integrases are unidirectional in the absence of accessory factors, phage integrases can be used for site-specific genome manipulations and for the stable integration of transgenes ( 4 ). The integrase from the Streptomyces temperate phage fC31 is currently the most widely used phage integrase for genome manipulations ( 5?14 ). The fC31 integrase is a member of a large family of site-specific recombinases, the serine recombinases ( 15,16 ). The best understood members of this family are the resolvase/invertases, in particular gd and Tn3 resolvases and Hin invertase. The resolvase/invertases have an N-terminal catalytic domain ( 1?140 aa) and a small ( 141?183 aa), C-terminal DNA-binding domain. The fC31 integrase is a member of the large serine recombinases (17). These proteins have similar N-terminal catalytic domains to those of the resolvase/invertases, but they have much larger C-terminal domains of 300? 500 aa. The large serine recombinases also include some transposases such as TnpX from the Clostridium perfringens transposon, Tn4451, and TndX from the C. difficile conjugative transposon CTn5397 ( 18?20 ). Tn4451 and CTn5397 confer resistances to chloramphenicol and tetracycline, respectively. TnpX and TndX have been shown to be capable of integrating and excising their respective transposons with no requirement for any additional transposon-encoded accessory factors ( 20,21 ). The first step in a site-specific recombination pathway is recognition and binding of the substrates by the recombinase ( 22 ). Protein?protein interactions between the recombinase subunits then bring the two substrates together in a synaptic complex. Ghosh et al. ( 23 ) showed that dimers of mycobacteriophage Bxb1 integrase (a large serine recombinase) bind the substrates attB and attP, which are then probably brought together to form a synaptic tetramer. Post-synapsis, the resolvase/invertases and the large serine recombinases appear to use the same catalytic mechanism ( 16,22?27 ). DNA cleavage generates 2 bp staggered breaks in both substrates with the concomitant formation of transient covalent phosphoserine bonds between the recombinases and the recessed 50 ends. Strand exchange is thought to occur by 1808 rotation of two recombinase subunits bound to half sites relative to the other two subunits ( 28?30 ). Joining of the half sites to form recombinant products is inhibited when the two base overhangs at the staggered breaks cannot base pair due to mismatches ( 31,32 ). In the serine integras (...truncated)


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Rowley, Paul A., Smith, Matthew C. A., Younger, Ellen, Smith, Margaret C. M.. A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination, Nucleic Acids Research, 2008, pp. 3879-3891, Volume 36, Issue 12, DOI: 10.1093/nar/gkn269