Site-specific recombinases: molecular machines for the Genetic Revolution

Olorunniji, Femi J.; Rosser, Susan J.; Stark, W. Marshall

doi:10.1042/bj20151112

Cited by 79 publications

(78 citation statements)

References 145 publications

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“…The current view of the strand exchange process for the serine integrases suggests that it is overall approximately isoenergetic, and thus its complexities will not affect directionality. We also note here that ∼50% of PB–int s synapses are predicted to be incompetent for strand exchange because the two att sites are misaligned ‘in antiparallel’ (1,7,11,24,26). In these synapses, the two att sites must dissociate and reassociate to reach a strand exchange-proficient synapse.…”

Section: Resultsmentioning

confidence: 71%

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

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 71%

“…It is known that strand exchange is a complex process involving strand cleavages, subunit rotation and strand re-ligations, steps that are likely to be accompanied by protein conformational changes (1,24,25). However, for simplicity these are all condensed into a single step in our model.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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“…Whilst conditional GEMMs are a key resource, one limitation is the inability to model natural tumour evolution (early versus late events), because of the simultaneous rather than temporal activation of multiple genetic events. The principle of different site-specific recombinases (Olorunniji et al, 2016) to achieve sequential mutations in a time-dependent way is possible, although have not yet been widely applied.…”

Section: The Impact Of Technological Advances On Breast Cancer Modelsmentioning

confidence: 99%

“…MMTV - Erbb2 ; MMTV -PyMT) are used rather than more physiologically relevant GEMMs. As stromal influences are likely to impact differently on subtypes of breast cancer (Wallace et al, 2011), it is important to consider the use of alternative site-specific recombinases (Olorunniji et al, 2016) to direct the stromal gene modification (e.g. Flp/ Frt ), which would permit investigation with the many available Cre/ loxP GEMMs.…”

Section: Building a Toolkit Fit For The Futurementioning

confidence: 99%

In vivomodels in breast cancer research: progress, challenges and future directions

Holen

Speirs

Morrissey

et al. 2017

Disease Models &Amp; Mechanisms

139

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Research using animal model systems has been instrumental in delivering improved therapies for breast cancer, as well as in generating new insights into the mechanisms that underpin development of the disease. A large number of different models are now available, reflecting different types and stages of the disease; choosing which one to use depends on the specific research question(s) to be investigated. Based on presentations and discussions from leading experts who attended a recent workshop focused on in vivo models of breast cancer, this article provides a perspective on the many varied uses of these models in breast cancer research, their strengths, associated challenges and future directions. Among the questions discussed were: how well do models represent the different stages of human disease; how can we model the involvement of the human immune system and microenvironment in breast cancer; what are the appropriate models of metastatic disease; can we use models to carry out preclinical drug trials and identify pathways responsible for drug resistance; and what are the limitations of patient-derived xenograft models? We briefly outline the areas where the existing breast cancer models require improvement in light of the increased understanding of the disease process, reflecting the drive towards more personalised therapies and identification of mechanisms of drug resistance.

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“…Site-specific recombinase (SSR) systems are widely used as tools to rearrange, insert, remove, and join DNA with virtually no upper limit in size. For biotechnology purposes, this can include the insertion of exogenous DNA into chromosomes, the fusing of DNA molecules, or the construction of synthetic gene networks [1]. The tyrosine (Y-rec) and serine (S-rec) recombination families are named for the catalytic residue of their respective integrase (Int) protein.…”

Section: Introductionmentioning

confidence: 99%

An att site-based recombination reporter system for genome engineering and synthetic DNA assembly

et al. 2017

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BackgroundDirect manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla).ResultsThe HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested.ConclusionsThe HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

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Site-specific recombinases: molecular machines for the Genetic Revolution

Cited by 79 publications

References 145 publications

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

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

In vivomodels in breast cancer research: progress, challenges and future directions

An att site-based recombination reporter system for genome engineering and synthetic DNA assembly

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