Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown

Shin, H, Holland, A ORCID: 0000-0003-3258-9457, Alsaleh, A, Retiz, AD, Pigli, YZ, Taiwo-Aiyerin, OT, Peña Reyes, T, Bello, AJ ORCID: 0000-0002-6731-2047, Quan, J, Tang, W, Olorunniji, FJ ORCID: 0000-0001-9389-2981 and Rice, PA ORCID: 0000-0002-3467-341X (2025) Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown. Nucleic Acids Research, 53 (14). ISSN 0305-1048

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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.

Item Type:	Article
Uncontrolled Keywords:	Integrases; DNA Nucleotidyltransferases; Serine; Recombination, Genetic; Integrases; Recombination, Genetic; Serine; DNA Nucleotidyltransferases; 3101 Biochemistry and Cell Biology; 31 Biological Sciences; Genetics; Biotechnology; Cancer; Integrases; Recombination, Genetic; Serine; DNA Nucleotidyltransferases; 05 Environmental Sciences; 06 Biological Sciences; 08 Information and Computing Sciences; Developmental Biology; 31 Biological sciences; 34 Chemical sciences; 41 Environmental sciences
Subjects:	R Medicine > RS Pharmacy and materia medica
Divisions:	Pharmacy and Biomolecular Sciences
Publisher:	Oxford University Press
Date of acceptance:	18 July 2025
Date of first compliant Open Access:	1 September 2025
Date Deposited:	01 Sep 2025 10:57
Last Modified:	01 Sep 2025 11:00
DOI or ID number:	10.1093/nar/gkaf691
URI:	https://researchonline.ljmu.ac.uk/id/eprint/27036

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