DNA cleavage is independent of synapsis during Streptomyces phage phiBT1 integrase-mediated site-specific recombination.
Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.
Zhang
Lin
L
Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
Wang
Lu
L
Wang
Jin
J
Ou
Xijun
X
Zhao
Guoping
G
Ding
Xiaoming
X
D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D023902:Chromosome Pairing; D053837:DNA Cleavage; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration
D014764:Viral Proteins; D019426:Integrases