title: Engineering and characterisation of hybrid Large Serine Recombinases as tools for probing determinants of DNA sequence specificity
Creators: name: Lawson-Williams, M
Publisher: name: Liverpool John Moores University
Year: 2027

description: Phage-derived serine integrases are members of the large serine recombinase (LSRs) family of sitespecific recombinases that mediate highly precise DNA recombination. LSRs recombine pairs of short DNA sequences, referred to as attachment sites, attB and attP, by catalysing unidirectional double stranded DNA cleavage and rejoining reactions. The reaction products are newly formed recombination sites, attL and attR. In nature this process is required for the integration of phage DNA into their bacterial host genomes, where they remain as prophages. The reverse reaction, known as excision, is mediated by the same integrase protein in the presence of a second phage-encoded protein called the recombination directionality factor (RDF), to recombine attL and attR sites back to attP and attB. Knowledge of the rules that govern the strict specificity LSRs have for their cognate attachment sites is limited but is critical to the expansion of the application of LSRs in synthetic biology and genome engineering. This work utilises AlphaFold-predicted structural models and synthetic biology approaches to rationally design hybrid LSRs derived from naturally occurring integrases as tools for probing protein-DNA interactions that determine sequence recognition and catalysis. Hybrid integrases derived from ΦC31 and TG1 integrases catalyse recombination of similarly hybridised sites and maintain interactions with the recombination directionality factor cognate to the integrase region that mediates RDF interaction. The hybrids retain the ability to discriminate between sites, and only promote recombination between sequences that meet their specificity requirements. Analysis of the activities of hybrid integrases on different combinations of hybrid att sites highlights critical motifs involved in sequence recognition, namely the recombinase domain binding motif. The findings demonstrate a clear and effective strategy for designing modular, programmable integrases capable of targeting and recombining user defined att sites. Further, Shawty integrase, a novel LSR which is a homolog of TG1 integrase, was identified via bioinformatic search, cloned, purified, and characterised. Shawty integrase exhibits unique properties, including interaction with both ΦC31 integrase and TG1 integrase cognate RDFs. Purification and characterisation of Shawty integrase adds another valuable protein to the toolkit of highly active integrases available for synthetic biology applications. Comparative analyses of the activities of Shawty integrase and TG1 integrase on hybrid sites provided additional insights into protein domains that control attB sequence recognition and suggests an alternative way to design synthetic recombinases. It is anticipated that availability of an expanded set of integrases through genome mining, further characterisation of the structural determinants of sequence specificity, and refinement of protein structure prediction tools will help facilitate better design of recombinases with novel specificities.