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bioRxiv · 10.1101/2025.05.02.651858

Structural basis of DNA recombination catalysis and regulation by phiC31 integrase

Sun, Y. E.·Aspinall, L. P.·Joseph, A. P.·Colloms, S. D.·Stark, M.·Spagnolo, L.
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Abstract

Large serine integrases catalyse the integration and excision of bacteriophage (phage) DNA genomes into and from the genomes of their bacterial hosts by site-specific recombination1. These recombination reactions are notable for their unidirectionality: integrase recognises and efficiently recombines short (40-50-bp) sequences in the phage (attP) and bacterial (attB) genomes, but is inactive on the product sites attL and attR flanking the inserted prophage, ensuring stable integration. The reverse reaction (excision) only occurs in the presence of a second phage-encoded protein, the Recombination Directionality Factor (RDF). Their strict recombination directionality has made serine integrases versatile tools in emerging genome-editing technologies2-5; however, the structural basis of directionality remains. Here we report structures of {phi}C31 integrase, the most-studied and most widely used member of the serine integrase family, in complexes with its DNA recombination sites, with and without its RDF. These structures correspond to four key mechanism steps: integration of the phage DNA genome (attP x attB); excision of the integrated prophage DNA, mediated by integrase and RDF (attL x attR); inhibition of integrase-catalysed excision in the absence of RDF (attL dimer complex); and inhibition of integrase-catalysed integration in the presence of RDF (attB dimer complex). Our data provide a mechanistic understanding of how the serine integrase recombination system establishes and regulates phage lysogeny, and lay the foundation for future development of integrase-based genome-editing technologies.

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Sun, Y. E., Aspinall, L. P., Joseph, A. P., Colloms, S. D., Stark, M., Spagnolo, L.. 2025-05-02. Structural basis of DNA recombination catalysis and regulation by phiC31 integrase. https://doi.org/10.1101/2025.05.02.651858

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