Sequences in attB that affect the ability of phiC31 integrase to synapse and to activate DNA cleavage.
Phage integrases are required for recombination of the phage genome with the host chromosome either to establish or exit from the lysogenic state. C31 integrase is a member of the serine recombinase family of site-specific recombinases. In the absence of any accessory factors integrase is unidirectional, catalysing the integration reaction between the phage and host attachment sites, attP x attB to generate the hybrid sites, attL and attR. The basis for this directionality is due to selective synapsis of attP and attB sites. Here we show that mutations in attB can block the integration reaction at different stages. Mutations at positions distal to the crossover site inhibit recombination by destabilizing the synapse with attP without significantly affecting DNA-binding affinity. These data are consistent with the proposal that integrase adopts a specific conformation on binding to attB that permits synapsis with attP. Other attB mutants with changes close to the crossover site are able to form a stable synapse but cleavage of the substrates is prevented. These mutants indicate that there is a post-synaptic DNA recognition event that results in activation of DNA cleavage.
Gupta
Milind
M
Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
Till
Rob
R
Smith
Margaret C M
MC
D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D019426:Integrases / Q000378:metabolism*; D016296:Mutagenesis; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D019426:Integrases