Structural basis of directionality control in large serine integrases.

Large serine integrases catalyze unidirectional insertion of large DNA payloads and, in the presence of a cognate recombination directionality factor (RDF), catalyze unidirectional excision. Understanding how directionality is enforced has been hampered by a lack of structural information. Here, we report 8 structures of SPβ integrase-DNA complexes along the integrative (-RDF) and excisive (+RDF) reaction pathways at resolutions extending to 3.15 Å. These include tetrameric intermediates before and after strand exchange and product-bound dimers for both pathways. We find that recombination-induced conformational changes and RDF-mediated repositioning of the integrase's coiled-coil subdomain (1) dictate which pairs of DNA sites can be assembled into active synaptic complexes and (2) dictate which product complexes will be conformationally locked, preventing back reactions. Critically, we find that the synaptic complex in which excision occurs is fundamentally different from that in which integration occurs. These insights provide a framework for engineering improved genome editing tools.