)
| Restriction endonuclease EcoRV | |||||||||
|---|---|---|---|---|---|---|---|---|---|
) EcoRV crystal structure complexed with double-stranded DNA | |||||||||
| Identifiers | |||||||||
| Symbol | Endonuc-EcoRV | ||||||||
| Pfam | PF09233 | ||||||||
| InterPro | IPR015314 | ||||||||
| SCOP2 | 1sx5 / SCOPe / SUPFAM | ||||||||
| |||||||||
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.
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 DA, Aggarwal AK, Schildkraut I (1998). "FokI dimerization is required for DNA cleavage". Proc Natl Acad Sci USA. 95 (18): 10570–10575. Bibcode:1998PNAS...9510570B. 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.