DNA cleavage is independent of synapsis during streptomyces phage Φ BT1 integrase-mediated site-specific recombination

Abstract

Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of Φ BT1 and Φ C31 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. © The Author (2010).