DNA Cleavage is Independent of Synapsis during Streptomyces Phage φBT1 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 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.

Streptomyces temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms)

“…The integration process was very eYcient with attB and attP substrates, but was also measurable with attL and attR sequences, implying that Int might excise BT1 in vivo at some frequency in the absence of other factors. Further mechanistic studies have been reported recently [107]. Although no studies have been carried out to characterize an Xis or RDF protein, BLASTP analysis with the 244-aa gp3 RDF from C31 gave a top hit of 84.8% amino acid identities to a 247-aa gp3 protein from BT1 (this report).…”

Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces

“…In addition to the intact attP - attB pair, there are 15 mutated attP - attB pairs which can be recognized by ϕBT1 integrase17. Multiple rounds of large fragment deletion can be achieved with the following modifications.…”

“…Both ϕC31 and ϕBT1 attP - int loci have been used to construct versatile vectors which can integrate into different attB sites in Streptomyces 1516. To increase the diversity of attP - attB pair of ϕBT1, 15 mutated attP - attB pairs ( attP 01 - attB 01 → attP 15 - attB 15 ) were generated by PCR mutagenesis of the central dinucleotide sequence of attB and attP 17. The Cre/loxP system was successfully used for the deletion of large fragments in Magnetospirillum gryphiswaldense and several Streptomyces species181920.…”

Mechanistic Modeling of a Rewritable Recombinase Addressable Data Module

“…Monomeric integrase is known to form dimers reversibly in solution [11], [13]- [36], and thus we can write the biochemical equation…”

“…Binding of gp3 to dimeric integrase already bound to both attL and attR is necessary to mediate excision, restoring attB and attP [11], [13], [17], [20]- [23], [25], [26], [28], [30], [36], [40], [42], [44], [45], [47], [49], [54] …”