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Highly efficient in vitro site-specific recombination system based on streptomyces phage phiBT1 integrase.

ABSTRACT: The Streptomyces phage phiBT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the phiBT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra- and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the phiBT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.

SUBMITTER: Zhang L

PROVIDER: S-EPMC2565996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Control of directionality in Streptomyces phage ?BT1 integrase-mediated site-specific recombination.

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Project description:Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo. However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is dictated by the increasing complexity of the genetic studies. Here, we describe a novel site-specific recombination system designated Vika/vox. Vika originates from a degenerate bacteriophage of Vibrio coralliilyticus and shares low sequence similarity to other tyrosine recombinases, but functionally carries out a similar type of reaction. We demonstrate that Vika is highly specific in catalyzing vox recombination without recombining target sites from other SSR systems. We also compare the recombination activity of Vika/vox with other SSR systems, providing a guideline for deciding on the most suitable enzyme for a particular application and demonstrate that Vika expression does not cause cytotoxicity in mammalian cells. Our results show that Vika/vox is a novel powerful and safe instrument in the 'genetic toolbox' that can be used alone or in combination with other SSRs in heterologous hosts.

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Project description:Phage-encoded serine integrases mediate directionally regulated site-specific recombination between short attP and attB DNA sites without host factor requirements. These features make them attractive for genome engineering and synthetic genetics, although the basis for DNA site selection is poorly understood. Here we show that attP selection is determined through multiple proofreading steps that reject non-attP substrates, and that discrimination of attP and attB involves two critical site features: the outermost 5-6 base pairs of attP that are required for Int binding and recombination but antagonize attB function, and the "discriminators" at positions -15/+15 that determine attB identity but also antagonize attP function. Thus, although the attachment sites differ in length and sequence, only two base changes are needed to convert attP to attL, and just two more from attL to attB. The opposing effect of site identifiers ensures that site schizophrenia with dual identities does not occur.

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