Attachment Site Selection and Identity in Bxb1 Serine Integrase-Mediated Site-Specific Recombination

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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.

Vyšlo v časopise: Attachment Site Selection and Identity in Bxb1 Serine Integrase-Mediated Site-Specific Recombination. PLoS Genet 9(5): e32767. doi:10.1371/journal.pgen.1003490
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003490

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Zdroje

1. LandyA (1989) Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem 58: 913–949.

2. GrindleyND, WhitesonKL, RicePA (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75: 567–605.

3. LewisJA, HatfullGF (2001) Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins. Nucleic Acids Res 29: 2205–2216.

4. LandyA (1993) Mechanistic and structural complexity in the site-specific recombination pathways of Int and FLP. Curr Opin Genet Dev 3: 699–707.

5. SmithMC, ThorpeHM (2002) Diversity in the serine recombinases. Mol Microbiol 44: 299–307.

6. KhaleelT, YoungerE, McEwanAR, VargheseAS, SmithMC (2011) A phage protein that binds phiC31 integrase to switch its directionality. Mol Microbiol 80: 1450–1463.

7. GhoshP, WasilLR, HatfullGF (2006) Control of Phage Bxb1 Excision by a Novel Recombination Directionality Factor. PLoS Biol 4: e186 doi:10.1371/journal.pbio.0040186.

8. HillmanRT, CalosMP (2012) Site-specific integration with bacteriophage PhiC31 integrase. Cold Spring Harb Protoc 2012

9. NkrumahLJ, MuhleRA, MouraPA, GhoshP, HatfullGF, et al. (2006) Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nat Methods 3: 615–621.

10. ChavezCL, KeravalaA, WoodardLE, HillmanRT, StoweTR, et al. (2010) Kinetics and longevity of PhiC31 integrase in mouse liver and cultured cells. Hum Gene Ther 21: 1287–1297.

11. HuangJ, GhoshP, HatfullGF, HongY (2011) Successive and targeted DNA integrations in the Drosophila genome by Bxb1 and phiC31 integrases. Genetics 189: 391–395.

12. BonnetJ, SubsoontornP, EndyD (2012) Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc Natl Acad Sci U S A

13. FriedlandAE, LuTK, WangX, ShiD, ChurchG, et al. (2009) Synthetic gene networks that count. Science 324: 1199–1202.

14. KimAI, GhoshP, AaronMA, BibbLA, JainS, et al. (2003) Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Mol Microbiol 50: 463–473.

15. GhoshP, KimAI, HatfullGF (2003) The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. Mol Cell 12: 1101–1111.

16. BaiH, SunM, GhoshP, HatfullGF, GrindleyND, et al. (2011) Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase. Proc Natl Acad Sci U S A 108: 7419–7424.

17. GhoshP, PannunzioNR, HatfullGF (2005) Synapsis in phage Bxb1 integration: selection mechanism for the correct pair of recombination sites. J Mol Biol 349: 331–348.

18. ThorpeHM, SmithMC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 95: 5505–5510.

19. ThorpeHM, WilsonSE, SmithMC (2000) Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31. Mol Microbiol 38: 232–241.

20. SmithMA, TillR, SmithMC (2004) Switching the polarity of a bacteriophage integration system. Mol Microbiol 51: 1719–1728.

21. MoritaK, YamamotoT, FusadaN, KomatsuM, IkedaH, et al. (2009) In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase. Mol Genet Genomics 282: 607–616.

22. MandaliS, DharG, AvliyakulovNK, HaykinsonMJ, JohnsonRC (2013) The site-specific integration reaction of Listeria phage A118 integrase, a serine recombinase. Mob DNA 4: 2.

23. ZhangL, OuX, ZhaoG, DingX (2008) Highly efficient in vitro site-specific recombination system based on streptomyces phage phiBT1 integrase. J Bacteriol 190: 6392–6397.

24. BreunerA, BrondstedL, HammerK (2001) Resolvase-like recombination performed by the TP901-1 integrase. Microbiology 147: 2051–2063.

25. GuptaM, TillR, SmithMC (2007) Sequences in attB that affect the ability of phiC31 integrase to synapse and to activate DNA cleavage. Nucleic Acids Res 35: 3407–3419.

26. GhoshP, BibbLA, HatfullGF (2008) Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading. Proc Natl Acad Sci U S A 105: 3238–3243.

27. SmithMA, TillR, BradyK, SoultanasP, ThorpeH, et al. (2004) Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination. Nucleic Acids Res 32: 2607–2617.

28. SmithMC, BrownWR, McEwanAR, RowleyPA (2010) Site-specific recombination by phiC31 integrase and other large serine recombinases. Biochem Soc Trans 38: 388–394.

29. GrothAC, OlivaresEC, ThyagarajanB, CalosMP (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci U S A 97: 5995–6000.

30. ChavezCL, CalosMP (2011) Therapeutic applications of the PhiC31 integrase system. Curr Gene Ther 11: 375–381.

31. KeravalaA, GrothAC, JarrahianS, ThyagarajanB, HoytJJ, et al. (2006) A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Mol Genet Genomics 276: 135–146.