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Abstract Integrases from the “large serine” family are simple, highly directional site-specific DNA recombinases that have great promise as synthetic biology and genome editing tools. Integrative recombination (mimicking phage or mobile element insertion) requires only integrase and two short (∼40–50) DNA sites. The reverse reaction, excisive recombination, does not occur until it is triggered by the presence of a second protein termed a recombination directionality factor (RDF), which binds specifically to its cognate integrase. Identification of RDFs has been hampered due to their lack of sequence conservation and lack of synteny with the phage integrase gene. Here we use AlphaFold2-multimer to identify putative RDFs for more than half of a test set of 98 large serine recombinases, and experimental methods to verify predicted RDFs for 6 of 9 integrases chosen as test cases. We find no universally conserved structural motifs among known and predicted RDFs, yet they are all predicted to bind a similar location on their cognate integrase, suggesting convergent evolution of function. Our methodology greatly expands the available genetic toolkit of cognate integrase–RDF pairs. 

Abstract Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favour excision over insertion. Although RDFs are predicted to bind their cognate LSIs in similar ways, there is no overall common structural theme across LSI RDFs, leading to the suggestion that some of them may be moonlighting proteins with other primary functions. To test this hypothesis, we searched for characterized proteins with structures similar to the predicted structures of known RDFs. Our search shows that the RDFs for two LSIs, TG1 integrase and Bxb1 integrase, show high similarities to a single-stranded DNA binding (SSB) protein and an editing exonuclease, respectively. We present experimental data to show that Bxb1 RDF is probably an exonuclease and TG1 RDF is a functional SSB protein. We used mutational analysis to validate the integrase-RDF interface predicted by AlphaFold2 multimer for TG1 integrase and its RDF, and establish that control of recombination directionality is mediated via protein–protein interaction at the junction of recombinase’s second DNA binding domain and the base of the coiled-coil domain.