''Eco''RV (pronounced "eco R five") is a type II restriction
endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...
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 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.
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 modific ...
, EcoRV forms a
homodimer in solution before binding and acting on its
recognition sequence.
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
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...
known to cause a major protein-induced conformational change in the DNA.
Uses
EcoRV is often used to cut open a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
vector to insert a
gene-of-interest during
gene cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
. 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 modific ...
, another nuclease enzyme from ''E. coli''.
*
EcoRII, 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. O ...
, 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