In vitro properties of large serine integrase hybrids derived from ϕC31 and TG1 integrases

Lawson-Williams, M, Holland, A, Bello, AJ ORCID: 0000-0002-6731-2047, Rice, PA ORCID: 0000-0002-3467-341X and Olorunniji, FJ ORCID: 0000-0001-9389-2981 (2026) In vitro properties of large serine integrase hybrids derived from ϕC31 and TG1 integrases. Nucleic Acids Research, 54 (14). ISSN 0305-1048

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Abstract

Phage-derived serine integrases are site-specific recombinases from the large serine recombinase (LSR) family that catalyse precise DNA rearrangements. Their high specificity and unidirectional activity make them attractive tools for genome engineering and synthetic biology. However, their strict sequence requirements limit application to predefined target sites. Here, we report in vitro activities of hybrid integrases derived from ϕC31 and TG1 integrases that efficiently recombine hybrid attP × attB substrates while preserving interaction with the cognate recombination directionality factor (RDF) via the TG1-derived coiled-coil domain. These hybrid recombinases retain site discrimination and catalyse attL × attR recombination exclusively in the presence of the appropriate RDF, indicating preserved directionality control. Analysis of the activities of hybrid integrases across a panel of hybrid substrates highlight the importance of DNA-binding domains-att site recognition motifs in governing specificity. This study presents a modular framework for engineering programmable serine integrases with tailored specificity. Combined with structural prediction tools and expanded LSR discovery and characterization, this strategy holds promise for rational design of recombinases targeting custom genomic sites.

Item Type:	Article
Uncontrolled Keywords:	Siphoviridae; Bacteriophages; Integrases; DNA Nucleotidyltransferases; Serine; DNA; Recombination, Genetic; Substrate Specificity; Models, Molecular; Attachment Sites, Microbiological; Integrases; Recombination, Genetic; Substrate Specificity; Attachment Sites, Microbiological; Bacteriophages; Serine; DNA Nucleotidyltransferases; Models, Molecular; DNA; Siphoviridae; 3101 Biochemistry and Cell Biology; 31 Biological Sciences; Genetics; Biotechnology; Integrases; Recombination, Genetic; Substrate Specificity; Attachment Sites, Microbiological; Bacteriophages; Serine; DNA Nucleotidyltransferases; Models, Molecular; DNA; Siphoviridae; 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:	7 July 2026
Date of first compliant Open Access:	16 September 2026
Date Deposited:	16 Sep 2026 10:20
Last Modified:	16 Sep 2026 10:20
DOI or ID number:	10.1093/nar/gkag750
URI:	https://researchonline.ljmu.ac.uk/id/eprint/29450

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