Английская Википедия:FokI

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Шаблон:Short description

Шаблон:Infobox protein family

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.[1] 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,[2] 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.[2]

DNA-cleavage domain

DNA cleavage is mediated through the non-specific cleavage domain which also includes the dimerisation surface.[3] The dimer interface is formed by the parallel helices α4 and α5 and two loops P1 and P2 of the cleavage domain.[2]

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.[3]

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),[1] or inactive Cas9[4][5][6]

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.[7]

References

  1. ↑ 1,0 1,1 Шаблон:Cite journal
  2. ↑ 2,0 2,1 2,2 Шаблон:Cite journal
  3. ↑ 3,0 3,1 Шаблон:Cite journal
  4. ↑ Tsai, S. Q. et al. (2014). Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576 Шаблон:Doi
  5. ↑ 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 Шаблон:Doi
  6. ↑ 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 Шаблон:Doi
  7. ↑ Шаблон:Cite journal

See also

Шаблон:Restriction enzyme

Партнерские ресурсы
Криптовалюты
Магазины
Хостинг
Разное

Источник — http://wikihandbk.com/ruwiki/index.php?title=Английская_Википедия:FokI&oldid=14731838