The mechanism of ϕC31 integrase directionality: experimental analysis and computational modelling.

Alexandra Pokhilko,Oliver Ebenhöh,Jia Zhao,Margaret C M Smith,Sean D Colloms,W Marshall Stark

doi:10.1093/nar/gkw616

Abstract

Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant attL and attR sites, whereas the ‘reverse’ reaction (between attL and attR) requires an additional protein, the recombination directionality factor (RDF). Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ϕC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase–DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination.

Highlights

The serine integrases are a group of DNA site-specific recombinases whose natural functions are to integrate and excise bacteriophage DNA into and out from the host bacterial genomic DNA [1]
Published data on the kinetics of ␾C31 integrase-mediated reactions have been obtained under a variety of experimental conditions, and using different kinds of DNA substrates
Two previous works have modelled the kinetics of recombination by serine integrases [8,32], but both studies make mechanistic assumptions that do not accord with experimental observations

Summary

Introduction

The serine integrases are a group of DNA site-specific recombinases whose natural functions are to integrate and excise bacteriophage DNA into and out from the host bacterial genomic DNA [1]. The subject of this report is ␾C31 integrase, perhaps the most extensively studied member of the group, which has been exploited for applications including integrating vectors for bacteria, gene therapy in mammalian cells, gene knock-in/knock-out in various experimental organisms, gene/metabolic pathway assembly, genetic switches, logic gates and memory devices [2,3,4,5,6,7,8,9]. The ‘reverse’ reaction between attL and attR (L × R) is not observed in the presence of the integrase alone. The presence of a recombination directionality factor (RDF) protein transforms the activity of integrase so that it promotes L × R recombination, leading to attP and attB products [1,10,11,12,13]

Methods

Results

Conclusion