Biology:FokI
| Restriction endonuclease Fok1, C terminal | |||||||||
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| Identifiers | |||||||||
| Symbol | Endonuc-Fok1_C | ||||||||
| Pfam | PF09254 | ||||||||
| Pfam clan | CL0415 | ||||||||
| InterPro | IPR015334 | ||||||||
| SCOP2 | 2fok / SCOPe / SUPFAM | ||||||||
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The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non sequence-specific DNA cleavage domain at the C-terminal.[2] Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves the DNA at two locations, regardless of the nucleotide sequence at the cut site. The DNA is cut 9 nucleotides downstream of the motif on the forward strand, and 13 nucleotides downstream of the motif on the reverse strand,[3] producing two sticky ends with 4-bp overhangs.
Its molecular mass is 65.4 kDa, being composed of 587 amino acids.
DNA-binding domain
The recognition domain contains three subdomains (D1, D2 and D3) that are evolutionarily related to the DNA-binding domain of the catabolite gene activator protein which contains a helix-turn-helix.[3]
DNA-cleavage domain
DNA cleavage is mediated through the non-specific cleavage domain which also includes the dimerisation surface.[4] The dimer interface is formed by the parallel helices α4 and α5 and two loops P1 and P2 of the cleavage domain.[3]
Activity
When the nuclease is unbound to DNA, the endonuclease domain is sequestered by the DNA-binding domain and is released through a conformational change in the DNA-binding domain upon binding to its recognition site. Cleavage only occurs upon dimerization, when the recognition domain is bound to its cognate site and in the presence of magnesium ions.[4]
Exploitation
The endonuclease domain of Fok1 has been used in several studies, after combination with a variety of DNA-binding domains such as the zinc finger (see zinc finger nuclease),[2] or inactive Cas9[5][6][7]
One of several human vitamin D receptor gene variants is caused by a single nucleotide polymorphism in the start codon of the gene which can be distinguished through the use of the Fok1 enzyme.[8]
References
- ↑ Aggarwal, A. K.; Wah, D. A.; Hirsch, J. A.; Dorner, L. F.; Schildkraut, I. (1997). "Structure of the multimodular endonuclease Fok1 bound to DNA". Nature 388 (6637): 97–100. doi:10.1038/40446. PMID 9214510.
- ↑ 2.0 2.1 "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells". Nucleic Acids Res 33 (18): 5978–90. 2005. doi:10.1093/nar/gki912. PMID 16251401.
- ↑ 3.0 3.1 3.2 Wah, D. A.; Bitinaite, J.; Schildkraut, I.; Aggarwal, A. K. (1998). "Structure of Fok1 has implications for DNA cleavage". Proc Natl Acad Sci USA 95 (18): 10564–9. doi:10.1073/pnas.95.18.10564. PMID 9724743. Bibcode: 1998PNAS...9510564W.
- ↑ 4.0 4.1 Bitinaite, J.; Wah, D. A.; Aggarwal, A. K.; Schildkraut, I. (1998). "Fok1 dimerization is required for DNA cleavage". Proc Natl Acad Sci USA 95 (18): 10570–5. doi:10.1073/pnas.95.18.10570. PMID 9724744. Bibcode: 1998PNAS...9510570B.
- ↑ Tsai, S. Q. et al. (2014). Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576 doi:10.1038/nbt.2908
- ↑ Guilinger, J. P., Thompson, D. B. & Liu, D. R. (2014). Fusion of catalytically inactive Cas9 to Fok1 nuclease improves the specificity of genome modification. Nature Biotechnol. 32, 577–582 doi:10.1038/nbt.2909
- ↑ Wyvekens, N., Topkar, V. V., Khayter, C., Joung, J. K. & Tsai, S. Q. (2015). Dimeric CRISPR RNA-guided Fok1-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431 doi:10.1089/hum.2015.084
- ↑ Strandberg, S. (2003). "Vitamin D receptor start codon polymorphism (Fok1) is related to bone mineral density in healthy adolescent boys". J Bone Miner Metab 21 (2): 109–13. doi:10.1007/s007740300018. PMID 12601576.
See also
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