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bioRxiv · 10.1101/2024.08.21.608926

Large serine integrases utilise scavenged phage proteins as directionality cofactors

Alsaleh, A.·Pena Reyes, T.·Baksh, A.·Taiwo-Aiyerin, O. T.·Holland, A.·Pigli, Y. Z.·Rice, P. A.·Olorunniji, F. J.
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Abstract

Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favor 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 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 recombinases second DNA binding domain and the base of the coiled coil domain.

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Alsaleh, A., Pena Reyes, T., Baksh, A., Taiwo-Aiyerin, O. T., Holland, A., Pigli, Y. Z., Rice, P. A., Olorunniji, F. J.. 2024-08-21. Large serine integrases utilise scavenged phage proteins as directionality cofactors. https://doi.org/10.1101/2024.08.21.608926

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