- 関
- EcoRV・DNA複合体
認識配列
gatatc
切断部位
gat|atc
参考
- http://rebase.neb.com/rebase/enz/EcoRV.html
Wikipedia preview
出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/03/27 04:59:39」(JST)
[Wiki ja表示]
EcoRV（えこあーるふぁいぶ）は、平滑末端を作り出すII型の制限酵素の代表的なものである。大腸菌（Escherichia coli）のR株から見つかった5番目の制限酵素である。
作用
遺伝子中の 5'-GATATC-3' という6塩基配列を認識し、TとAの間に切れ目を入れ、切り口に平滑末端を作り出す。
EcoRVの切断パターン
切断前の配列 切断後の配列
5'-GATATC-3' 5'-GAT ATC-3'
3'-CTATAG-5' 3'-CTA TAG-5'
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この項目は、生物学に関連した書きかけの項目です。この項目を加筆・訂正などしてくださる協力者を求めています（プロジェクト:生命科学／Portal:生物学）。 |
[Wiki en表示]
DNA recognition sequence of EcoRV. The green line represents the cut site.
EcoRV cleaving DNA. The protein loosely binds DNA and scans for its recognition sequence. Once found, EcoRV kinks the DNA in a 50° angle and cleaves at the cognate sequence.
| Restriction endonuclease EcoRV |
|
EcoRV crystal structure complexed with double-stranded DNA
|
| Identifiers |
| Symbol |
Endonuc-EcoRV |
| Pfam |
PF09233 |
| InterPro |
IPR015314 |
| SCOP |
1sx5 |
| SUPERFAMILY |
1sx5 |
| Available protein structures: |
| Pfam |
structures |
| PDB |
RCSB PDB; PDBe; PDBj |
| PDBsum |
structure summary |
|
EcoRV (pronounced "eco R five") is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I.
In molecular biology, it is a commonly used restriction enzyme. It creates blunt ends. The enzyme recognizes the palindromic 6-base DNA sequence 5'-GAT|ATC-3' and makes a cut at the vertical line. The complementary sequence is then 3'-CTA|TAG-5'. The ends are blunt and can be ligated into a blunt cloning site easily but with lower efficiency than sticky ends.
Contents
- 1 Structure
- 2 Mode of action
- 3 Uses
- 4 See also
Structure
The structure of this enzyme, and several mutants, in complex with the DNA sequence which it cuts has been solved by X-ray crystallography.
The core of the enzyme consists of a five-stranded mixed β-sheet flanked by α-helices. The core is conserved in all other type II restriction endonucleases. It also has an N-terminal dimerization subdomain formed by a short α-helix, a two-stranded antiparallel -sheet, and a long α-helix. This subdomain is found only in EcoRV and PvuII. [1]
Mode of action
Like EcoRI, EcoRV forms a homodimer in solution before binding and acting on its recognition sequence. [2] Initially the enzyme binds weakly to a non-specific site on the DNA. It randomly walks along the molecule until the specific recognition site is found. [1] EcoRV has a high specificity for its target DNA sequence.
Binding of the enzyme induces a conformational change in the DNA, bending it by about 50°. DNA bending results in the unstacking of the bases, widening of the minor groove, and compression of the major groove. This brings the phosphodiester linkage to be broken closer to the active site of the enzyme, where it can be cleaved. Cleavage occurs within the recognition sequence, and does not require ATP hydrolysis.[1]
EcoRV is the only type II restriction endonuclease known to cause a major protein-induced conformational change in the DNA. [1]
Uses
EcoRV is often used to cut open a plasmid vector to insert a gene-of-interest during gene cloning. The enzyme is supplied by many manufacturers and requires bovine serum albumin to work properly.
See also
- EcoRI, another nuclease enzyme from E. coli.
- EcoRII, another nuclease enzyme from E. coli.
- FokI, a nuclease enzyme from Flavobacterium okeanokoites
[3]== References ==
- ^ a b c d Pingoud A, Jeltsch A (2001). "Structure and function of type II restriction endonucleases". Nucleic Acids Research 29 (18): 3705–3727. doi:10.1093/nar/29.18.3705. PMC 55916. PMID 11557805.
- ^ Bitinaite J, Wah D A, Aggarwal A K, Schildkraut I (1998). "FokI dimerization is required for DNA cleavage". Proc Natl Acad Sci USA 95 (18): 10570–10575. doi:10.1073/pnas.95.18.10570. PMC 27935. PMID 9724744.
- ^ Zahran, M., Daidone, I., Smith, J. C., & Imhof, P. (2010). Mechanism of DNA recognition by the restriction enzyme EcoRV. Journal of molecular biology, 401(3), 415-432.
English Journal
- The Impact of Five VDR Polymorphisms on Multiple Sclerosis Risk and Progression: a Case-Control and Genotype-Phenotype Study.
- Křenek P1, Benešová Y2, Bienertová-Vašků J1, Vašků A1.
- Journal of molecular neuroscience : MN.J Mol Neurosci.2018 Apr;64(4):559-566. doi: 10.1007/s12031-018-1034-1. Epub 2018 Mar 28.
- PMID 29589202
- Orthogonal Probing of Single-Molecule Heterogeneity by Correlative Fluorescence and Force Microscopy.
- Frederickx W1, Rocha S1, Fujita Y1, Kennes K1, De Keersmaecker H1, De Feyter S1, Uji-I H1,2, Vanderlinden W1,3.
- ACS nano.ACS Nano.2018 Jan 23;12(1):168-177. doi: 10.1021/acsnano.7b05405. Epub 2018 Jan 5.
- PMID 29257876
- Establishment of a Multiplex PCR-Based Procedure for Detection of Most Common Mutations in NPM1, FLT3 in Acute Myeloid Leukemia Patients.
- Trinh PL1, Pham Y2.
- Annals of clinical and laboratory science.Ann Clin Lab Sci.2018 Jan;48(1):35-39.
- PMID 29530994
Japanese Journal
- 日本産クリタケ自然集団内のミトコンドリアDNAの変異性
- 日本きのこ学会誌 : mushroom science and biotechnology 17(2), 65-69, 2009-07-31
- NAID 110007811856
- Identification of three Asian Hediste species (Polychaeta : Nereididae) by PCR-RFLP analysis of the mitochondrial 16S rRNA gene
- Monitoring of EcoRV Digestions on a DNA-immobilized Quartz Crystal Microbalance
Related Links
- ニュー・イングランド・バイオラボ －NEBは制限酵素、エンドヌクレアーゼ、リコンビナント酵素、PCR用試薬、発現システム、マーカー、コンピテントセル、RNA研究用試薬、ポリメラーゼ、修飾酵素、核酸、細胞解析などの試薬を提供 ...
- DNA塩基配列から各制限酵素認識配列を検索することができる簡単便利なWebツール【 Takara Cut-Site Navigator 】を公開しました。 制限酵素を使用される際に、ぜひ一度お試しください。
Related Pictures
