Structural basis of directionality control in large serine integrases

Large serine integrases (LSIs) catalyze unidirectional site-specific insertion of large DNA payloads, and in the presence of a cognate recombination directionality factor (RDF), catalyze unidirectional excision. Because neither reaction changes the net number of covalent bonds, the preferred direction must be controlled by the energetics of the changing protein-DNA complexes along these reaction pathways. However, a detailed understanding has been hampered by a lack of structural information. Here, we report 8 structures of SP{beta} integrase-DNA complexes along the integrative (-RDF) and excisive (+RDF) reaction pathways, at resolutions extending to 3.15 [A]. These complexes include tetrameric intermediates before and after strand exchange and product-bound dimers for both pathways. Our findings reveal that both recombination-induced conformational changes and RDF-mediated repositioning of the integrases coiled-coil subdomain (1) dictate which pairs of DNA sites can be assembled into a synaptic complex to initiate recombination 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 mechanistic insights provide a conceptual framework for engineering efficient and versatile genome editing tools.