A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase

Author:

Fan Hsiu-Fang123ORCID,Su Bo-Yu4,Ma Chien-Hui5,Rowley Paul A6ORCID,Jayaram Makkuni5ORCID

Affiliation:

1. Institute of Medical Science and Technology, National Sun Yat-sen University, Sizihwan, Kaohsiung 804, Taiwan

2. Department of Chemistry, National Sun Yat-sen University, Sizihwan, Kaohsiung 804, Taiwan

3. Aerosol Science Research Center, National Sun Yat-sen University, Sizihwan, Kaohsiung 804, Taiwan

4. Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan

5. Department of Molecular Biosciences, UT Austin, Austin, TX 78712, USA

6. Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA

Abstract

Abstract Streptomyces phage ϕC31 integrase (Int)—a large serine site-specific recombinase—is autonomous for phage integration (attP x attB recombination) but is dependent on the phage coded gp3, a recombination directionality factor (RDF), for prophage excision (attL x attR recombination). A previously described activating mutation, E449K, induces Int to perform attL x attR recombination in the absence of gp3, albeit with lower efficiency. E449K has no adverse effect on the competence of Int for attP x attB recombination. Int(E449K) resembles Int in gp3 mediated stimulation of attL x attR recombination and inhibition of attP x attB recombination. Using single-molecule analyses, we examined the mechanism by which E449K activates Int for gp3-independent attL x attR recombination. The contribution of E449K is both thermodynamic and kinetic. First, the mutation modulates the relative abundance of Int bound attL-attR site complexes, favoring pre-synaptic (PS) complexes over non-productively bound complexes. Roughly half of the synaptic complexes formed from Int(E449K) pre-synaptic complexes are recombination competent. By contrast, Int yields only inactive synapses. Second, E449K accelerates the dissociation of non-productively bound complexes and inactive synaptic complexes formed by Int. The extra opportunities afforded to Int(E499K) in reattempting synapse formation enhances the probability of success at fruitful synapsis.

Funder

Ministry of Science and Technology

Welch Foundation

National Science Foundation

National Institutes of Health

Center for Modeling Complex Interactions, University of Idaho

Publisher

Oxford University Press (OUP)

Subject

Genetics

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