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

Heewhan Shin; Alexandria Holland; Abdulrazak Alsaleh; Alyssa D. Retiz; Ying Z. Pigli; Oluwateniola T. Taiwo-Aiyerin; Tania Peña Reyes; Adebayo J. Bello; Jialiang Quan; Weixin Tang; Femi J. Olorunniji; Phoebe A. Rice

doi:10.1093/nar/gkaf691

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

Heewhan Shin, Alexandria Holland, Abdulrazak Alsaleh, Alyssa D. Retiz, Ying Z. Pigli, Oluwateniola T. Taiwo-Aiyerin, Tania Peña Reyes, Adebayo J. Bello, Jialiang Quan, Weixin Tang, Femi J. Olorunniji, Phoebe A. Rice

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Article number	gkaf691
Number of pages	14
Journal	Nucleic Acids Research
Volume	53
Issue number	14
Early online date	24 Jul 2025
DOIs	https://doi.org/10.1093/nar/gkaf691
Publication status	Published - 12 Aug 2025

Bibliographical note

Publisher Copyright:
© 2025 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research.

Data Availability Statement

The data underlying this article have been deposited
in Mendeley (output of AlphaFold2-multimer); VirtualPulldown_predicted_structures_full_length_LSI
(https://data.mendeley.com/datasets/ky6ptcbr6g/1), VirtualPulldown_predicted_structures_DBD2 (https://data.
mendeley.com/datasets/x8b3zwn2pf/1), and Int1_34
(https://data.mendeley.com/datasets/8ktg473htr/1). Plasmids used for the in vivo assays are being deposited with
Addgene.

Acknowledgements

Author contributions: Heewhan Shin (Investigation [equal],
Methodology [equal], Software [lead], Writing—review &
editing [supporting]), Alexandria Holland (Investigation
[equal]), Abdulrazak Alsaleh (Investigation [equal]), Alyssa
D. Retiz (Investigation [equal]), Ying Z. Pigli (Investigation [equal]), Oluwateniola T. Taiwo-Aiyerin (Investigation
[equal]), Tania Peña Reyes (Investigation [equal]), Adebayo
J. Bello (Investigation [equal]), Jialiang Quan (Investigation
[supporting]), Weixin Tang (Investigation [supporting]), Femi
J. Olorunniji (Conceptualization [equal], Funding acquisition [equal], Methodology [equal], Writing—review & editing [lead]), and Phoebe A. Rice (Conceptualization [equal],
Funding acquisition [equal], Methodology [equal], Writing—
original draft [lead]).

Funding

This work was supported by the National Science Foundation and UK Research and Innovation (collaborative grants NSF/BIO 2223480 and UKRI/BBSRC BB/X012085/1 to P.A.R. and F.J.O.). Funding to pay the Open Access publication charges for this article was provided by the BBSRC BB/X012085/1.

ASJC Scopus subject areas

Genetics

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10.1093/nar/gkaf691Licence: CC BY-NC

Cite this

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. (2025). Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown. Nucleic Acids Research, 53(14), Article gkaf691. https://doi.org/10.1093/nar/gkaf691

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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.",

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AU - Pigli, Ying Z.

AU - Taiwo-Aiyerin, Oluwateniola T.

AU - Peña Reyes, Tania

AU - Bello, Adebayo J.

AU - Quan, Jialiang

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AU - Olorunniji, Femi J.

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

Abstract

Bibliographical note

Data Availability Statement

Acknowledgements

Funding

ASJC Scopus subject areas

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