Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown
Open accessPublished version of a preprint
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.
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Cites
Highly accurate protein structure prediction with AlphaFold
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Accurate structure prediction of biomolecular interactions with AlphaFold 3
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- Authors
- Shin, H., Holland, A., Alsaleh, A., Retiz, A. D., Pigli, Y. Z., Taiwo-Aiyerin, O. T., Peña Reyes, T., Bello, A. J., Quan, J., Tang, W., Olorunniji, F. J., & Rice, P. A.
- Affiliations
- University of Chicago (US)
- Liverpool John Moores University (GB)
- The University of Chicago Department of Biochemistry & Molecular Biology, , Chicago, IL 60637 ,
- Corresponding author
- Olorunniji, Femi J
- Rice, Phoebe A
- ORCID
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2025-07-19
- Volume
- 53
- Issue
- 14
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC12286644
- OpenAlex
- W4412649839
- Cited by
- 9
- References
- 4
- Citations by year
- 2026: 5 · 2025: 3 · 2023: 1
- FWCI
- 2.37
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Liverpool John Moores University (acceptedVersion)
- Funders
- National Science Foundation
- UK Research and Innovation (NSF/BIO 2223480, UKRI/BBSRC BB/X012085/1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- site-specific recombination, integrative recombination, protein-protein interaction prediction, AlphaFold2-Multimer, convergent evolution, genome editing tools, synthetic biology
- MeSH terms
- DNA Nucleotidyltransferases, Recombination, Genetic, Serine, Integrases
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