Abstract
Phage-encoded serine integrases mediate directionally regulated site-specific recombination between short attP and attB DNA sites without host factor requirements. These features make them attractive for genome engineering and synthetic genetics, although the basis for DNA site selection is poorly understood. Here we show that attP selection is determined through multiple proofreading steps that reject non-attP substrates, and that discrimination of attP and attB involves two critical site features: the outermost 5–6 base pairs of attP that are required for Int binding and recombination but antagonize attB function, and the “discriminators” at positions −15/+15 that determine attB identity but also antagonize attP function. Thus, although the attachment sites differ in length and sequence, only two base changes are needed to convert attP to attL, and just two more from attL to attB. The opposing effect of site identifiers ensures that site schizophrenia with dual identities does not occur.
Highlights
Establishment of lysogeny by temperate bacteriophages typically involves site-specific integration of the phage genome into the host chromosome
The Integrase encoded by mycobacteriophage Bxb1 is a member of the serine-recombinase family and catalyzes strand exchange between attP and attB, the attachment sites for the phage and bacterial host, respectively
We address the question of what sequences within attP are required for it to act as an attP site and identify the key sequence features that are required not just for Integrase binding and for synapsis and post-synapsis events
Summary
Establishment of lysogeny by temperate bacteriophages typically involves site-specific integration of the phage genome into the host chromosome. Serine-integrases use simple attachment sites (,50 bp), have no host factor requirements, and the RDF does not act through direct binding to DNA [5,6,7]. Because of these features, serine-integrases function well in heterologous systems, making them attractive for genome engineering in human, mouse, drosophila, and malarial cells [8,9,10,11], as well as powerful switches for synthetic genetic circuits and microbial data storage systems [12,13]
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