''Eco''RV (pronounced "eco R five") is a type II restriction
endonuclease isolated from certain strains of ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...
''. It has the alternative name Eco32I.
In
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
, it is a commonly used
restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
. It creates
blunt ends
DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...
. The enzyme recognizes the palindromic 6-base DNA sequence 5'-GAT, ATC-3' and makes a blunt end 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
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
.
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.
Mode of action
Like
EcoRI
''Eco''RI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species '' E. coli.'' It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modifi ...
, EcoRV forms a
homodimer
In biochemistry, a protein dimer is a macromolecular complex formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ''dimer'' has ...
in solution before binding and acting on its
recognition sequence
A recognition sequence is a DNA sequence to which a structural motif of a DNA-binding domain exhibits binding specificity. Recognition sequences are palindromes.
The transcription factor Sp1 for example, binds the sequences 5'-(G/T)GGGCGG(G/A)(G/ ...
.
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.
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.
EcoRV is the only type II restriction
endonuclease known to cause a major protein-induced conformational change in the DNA.
Uses
EcoRV is often used to cut open a
plasmid vector to insert a
gene
In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
-of-interest during
gene cloning. The enzyme is supplied by many manufacturers and requires
bovine serum albumin
Bovine serum albumin (BSA or "Fraction V") is a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments.
The nickname "Fraction V" refers to albumin being the fifth fraction of the origin ...
to work properly.
See also
*
EcoRI
''Eco''RI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species '' E. coli.'' It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modifi ...
, another nuclease enzyme from ''E. coli''.
*
EcoRII
Restriction endonuclease (REase) EcoRII (pronounced "eco R two") is an enzyme of restriction modification system (RM) naturally found in ''Escherichia coli'', a Gram-negative bacteria. Its molecular mass is 45.2 Atomic mass unit, kDa, being compos ...
, another nuclease enzyme from ''E. coli''.
*
FokI
The restriction endonuclease ''Fok''1, naturally found in ''Flavobacterium okeanokoites'', is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal. ...
, a nuclease enzyme from ''Flavobacterium okeanokoites''
[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.]
References
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EC 3.1.21
Molecular biology
Bacterial enzymes
Restriction enzymes
