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Site-specific recombinases as tools for heterologous gene integration

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Abstract

Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes.

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References

  • Abremski K, Gottesman S (1981) Xis-independent excisive recombination of bacteriophage lambda. J Mol Biol 153:67–78

    CAS  PubMed  Google Scholar 

  • Akopian A, He J, Boocock MR, Stark WM (2003) Chimeric recombinases with designed DNA sequence recognition. Proc Natl Acad Sci USA 100:8688–8691

    CAS  PubMed  PubMed Central  Google Scholar 

  • Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7:649–659

    CAS  PubMed  Google Scholar 

  • Araki K, Araki M, Yamamura K (1997) Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acids Res 25:868–872

    CAS  PubMed  PubMed Central  Google Scholar 

  • Arnold PH, Blake DG, Grindley NDF, Boocock MR, Stark WM (1999) Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity. EMBO J 18:1407–1414

    CAS  PubMed  PubMed Central  Google Scholar 

  • Austin S, Ziese M, Sternberg N (1981) A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25:729–736

    CAS  PubMed  Google Scholar 

  • Bibb LA, Hancox MI, Hatfull GF (2005) Integration and excision by the large serine recombinase ϕRv1 integrase. Mol Microbiol 55:1896–1910

    CAS  PubMed  Google Scholar 

  • Biswas T, Aihara H, Radman-Livaja M, Filman D, Landy A, Ellenberger T (2005) A structural basis for allosteric control of DNA recombination by λ integrase. Nature 435:1059–1066

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bouhassira EE, Westerman K, Leboulch P (1997) Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange. Blood 90:3332–3344

    CAS  PubMed  Google Scholar 

  • Breuner A, Brondsted L, Hammer K (1999) Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1. J Bacteriol 181:7291–7297

    CAS  PubMed  PubMed Central  Google Scholar 

  • Broach JR, Guarascio VR, Jayaram M (1982) Recombination within the yeast plasmid 2 μ circle is site-specific. Cell 29:227–234

    CAS  PubMed  Google Scholar 

  • Brown WR, Lee NC, Xu Z, Smith MC (2011) Serine recombinases as tools for genome engineering. Methods 53:372–379

    CAS  PubMed  Google Scholar 

  • Bushman W, Thompson JF, Vargas L, Landy A (1985) Control of directionality in lambda site-specific recombination. Science 230:906–911

    CAS  PubMed  PubMed Central  Google Scholar 

  • Campbell A (1962) Episomes. Adv Genet 11:101–145

    Google Scholar 

  • Campbell A (1992) Chromosomal insertion site for phages and plasmids. J Bacteriol 174:7495–7499

    CAS  PubMed  PubMed Central  Google Scholar 

  • Carrasco CD, Ramaswamy KS, Ramasubramanian TS, Golden JW (1994) Anabaena xisF gene encodes a developmentally regulated site-specific recombinase. Genes Dev 8:74–83

    CAS  PubMed  Google Scholar 

  • Christ N, Corona T, Droge P (2002) Site-specific recombination in eukaryotic cells mediated by mutant λ integrases: implications for synaptic complex formation and the reactivity of episomal DNA segments. J Mol Biol 319:305–314

    CAS  PubMed  Google Scholar 

  • Christiansen B, Brondfted L, Vogensen FK, Hammer K (1996) A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1. J Bacteriol 178:5164–5173

    CAS  PubMed  PubMed Central  Google Scholar 

  • Enquist LW, Kikuchi A, Weisberg RA (1979) The role of λ integrase in integration and excision. Cold Spring Harbor Symp Quant Biol 43:1115–1120

    CAS  PubMed  Google Scholar 

  • Ghosh K, Van Duyne GD (2002) Cre-loxP biochemistry. Methods 28:374–383

    CAS  PubMed  Google Scholar 

  • Ghosh P, Wasil LR, Hatfull GF (2006) Control of phage Bxb1 excision by a novel recombination directionality factor. PLos Biol 4:e186

    PubMed  PubMed Central  Google Scholar 

  • Gilbertson L (2003) Cre–lox recombination: Cre-active tools for plant biotechnology. Trends Biotechnol 21:550–555

    CAS  PubMed  Google Scholar 

  • Gordley RM, Gersbach CA, Barbas CF III (2009) Synthesis of programmable integrases. Proc Natl Acad Sci USA 106:5053–5058

    CAS  PubMed  PubMed Central  Google Scholar 

  • Groth AC, Calos MP (2004) Phage integrases: biology and applications. J Mol Biol 335:667–678

    CAS  PubMed  Google Scholar 

  • Harel-Levy G, Goltsman J, Tuby CN, Yagil E, Kolot M (2008) Human genomic site-specific recombination catalyzed by coliphage HK022 integrase. J Biotechnol 134:46–54

    Google Scholar 

  • Heichman KA, Johnson RC (1990) The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer. Science 249:511–517

    CAS  PubMed  Google Scholar 

  • Hirano N, Muroi T, Kihara Y, Kobayashi R, Takahashi H, Haruki M (2011) Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes. Appl Microbiol Biotechnol 89:1877–1884

    CAS  PubMed  Google Scholar 

  • Khaleel T, Younger E, McEwan AR, Varghese AS, Smith MC (2011) A phage protein that binds ϕC31 integrase to switch its directionality. Mol Microbiol. doi:https://doi.org/10.1111/j.1365-2958

  • Klippel A, Kanaar R, Kahmann R, Cozzarelli NR (1993) Analysis of strand exchange and DNA binding of enhancer-independent Gin recombinase mutants. EMBO J 12:1047–1057

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kuhstoss S, Rao RN (1991) Analysis of the integration function of the streptomycete bacteriophage ϕC31. J Mol Biol 222:897–908

    CAS  PubMed  Google Scholar 

  • Langer SJ, Ghafoori AP, Byrd M, Leinwand L (2002) A genetic screen identifies novel non-compatible loxP sites. Nucleic Acids Res 30:3067–3077

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li W, Kamtekar S, Xiong Y, Sarkis GJ, Grindley NDF, Steitz TA (2005) Structure of a synaptic γδ resolvase tetramer covalently linked to two cleaved DNAs. Science 309:1210–1215

    CAS  PubMed  Google Scholar 

  • Lieu PT, Machleidt T, Thyagarajan B, Fontes A, Frey E, Fuerstenau-Sharp M, Thompson DV, Swamilingiah GM, Derebail SS, Piper D, Chesnut JD (2009) Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases. J Biomol Screen 14:1207–1215

    CAS  PubMed  Google Scholar 

  • Loonstra A, Vooijs M, Beverloo HB, Al Allak B, van Drunen E, Kanaar E, Berns A, Jonkers J (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci USA 98:9209–9214

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lorbach E, Christ N, Schwikardi M, Droge P (2000) Site-specific recombination in human cells catalyzed by phage λ integrase mutants. J Mol Biol 296:1175–1181

    CAS  PubMed  Google Scholar 

  • Matsuura M, Noguchi T, Yamaguchi D, Aida T, Asayama M, Takahashi H, Shirai M (1996) The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome. J Bacteriol 178:3374–3376

    CAS  PubMed  PubMed Central  Google Scholar 

  • Olivares EC, Hollis RP, Calos MP (2001) Phage R4 integrase mediates site-specific integration in human cells. Gene 278:167–176

    CAS  PubMed  Google Scholar 

  • Radman-Livaja M, Biswas T, Ellenberger T, Landy A, Aihara H (2006) DNA arms do the legwork to ensure the directionality of λ site-specific recombination. Curr Opin Struct Biol 16:42–50

    CAS  PubMed  Google Scholar 

  • Rice PA, Mouw KW, Montano SP, Boocock MR, Rowland SJ, Stark WM (2010) Orchestrating serine resolvases. Biochem Soc Trans 38:384–387

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rowley PA, Smith MC, Younger E, Smith MC (2008) A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination. Nucleic Acids Res 36:3879–3891

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sato T, Samori Y, Kobayashi Y (1990) The cisA cistron of Bacillus subtilis sporulation gene spoIVC encodes a protein homologue to a site-specific recombinase. J Bacteriol 172:1092–1098

    CAS  PubMed  PubMed Central  Google Scholar 

  • Schebelle L, Wolf C, Stribl C, Javaheri T, Schnuetgen F, Ettinger A (2010) Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FIEx gene traps. Nucleic Acids Res 38:e106

    PubMed  PubMed Central  Google Scholar 

  • Silverman M, Simon M (1980) Phase variation: genetic analysis of switching mutants. Cell 19:845–854

    CAS  PubMed  Google Scholar 

  • Smith MCM, Thorpe HM (2002) Diversity in the serine recombinases. Mol Microbiol 44:299–307

    CAS  PubMed  Google Scholar 

  • Smith MCM, Brown WRA, McEwan AR, Rowley PA (2010) Site-specific recombination by ϕC31 integrase and other large serine recombinases. Biochem Soc Trans 38:388–394

    CAS  PubMed  Google Scholar 

  • Suzuki N, Inui M, Yukawa H (2007) Site-directed integration system using a combination of mutant lox sites for Corynebacterium glutamicum. Appl Microbiol Biotechnol 77:871–878

    CAS  PubMed  Google Scholar 

  • Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP (2001) Site-specific genomic integration in mammalian cells mediated by phage ϕC31 integrase. Mol Cell Biol 21:3926–3934

    CAS  PubMed  PubMed Central  Google Scholar 

  • Turan S, Galla M, Ernst E, Qiao J, Voelkel C, Schiedlmeier B, Zehe C, Bode J (2011) Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. J Mol Biol 407:193–221

    CAS  PubMed  Google Scholar 

  • Van de Putte P, Cramer S, Giphart-Gassler M (1980) Invertible DNA determines host specificity of bacteriophage Mu. Nature 286:218–222

    PubMed  Google Scholar 

  • Yamaichi Y, Niki H (2004) migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome. EMBO J 23:221–233

    CAS  PubMed  Google Scholar 

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Acknowledgments

This work is supported by the 22nd Kato Memorial Bioscience Foundation and, in part, by a Grant-in-Aid for Young Scientists (B) No. 22760612, a grant to promote advanced scientific research, the Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Research Grant for 2010 from the College of Engineering, Nihon University.

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Authors and Affiliations

  1. Department of Chemical Biology & Applied Chemistry, College of Engineering, Nihon University, 1 Nakagawara, Tokusada, Tamura-machi, Koriyama, Fukushima, 963-8642, Japan

    Nobutaka Hirano, Tetsurou Muroi & Mitsuru Haruki

  2. Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, 252-8510, Japan

    Hideo Takahashi

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  1. Nobutaka Hirano
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  2. Tetsurou Muroi
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  3. Hideo Takahashi
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  4. Mitsuru Haruki
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Correspondence to Nobutaka Hirano.

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Hirano, N., Muroi, T., Takahashi, H. et al. Site-specific recombinases as tools for heterologous gene integration. Appl Microbiol Biotechnol 92, 227–239 (2011). https://doi.org/10.1007/s00253-011-3519-5

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  • DOI: https://doi.org/10.1007/s00253-011-3519-5

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