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Updated: Jun 2, 2025

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow
Published on: June 14, 2017
Structural basis of directionality control in large serine integrases
Heewhan Shin1, Ying Pigli1, Tania Peña Reyes1
1Department of Biochemistry & Molecular Biology, The University of Chicago; Chicago IL, 60637, USA.
Abstract:
Large serine integrases (LSIs) catalyze unidirectional site-specific DNA recombination reactions, yet those reactions are reversed by the presence of a cognate recombination directionality factor (RDF). Mechanistic understanding of directionality control has been hampered by a lack of structural information. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of six SPbeta integrase-DNA complexes along the integrative (-RDF) and excisive (+RDF) reaction pathways, at 4.16-7.18Å resolution. Our findings reveal how RDF-mediated repositioning of an integrase subdomain (1) dictates which pairs of DNA sites can be assembled into a synaptic complex to initiate recombination and (2) dictates which product complexes will be conformationally locked, preventing the back reaction. These mechanistic insights provide a conceptual framework for engineering efficient and versatile genome editing tools.
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