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The Info List - EcoRV


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EcoRV
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'-GATATC-3' and makes a cut at the vertical line. The complementary sequence is then 3'-CTATAG-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 5 References

Structure[edit] 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
EcoRV
and PvuII.[1] Mode of action[edit] Like EcoRI, EcoRV
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
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
EcoRV
is the only type II restriction endonuclease known to cause a major protein-induced conformational change in the DNA.[1] Uses[edit] EcoRV
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[edit]

EcoRI, another nuclease enzyme from E. coli. EcoRII, another nuclease enzyme from E. coli. FokI, a nuclease enzyme from Flavobacterium okeanokoites[3]

References[edit]

^ 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. 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.

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Hydrolase: esterases (EC 3.1)

3.1.1: Carboxylic ester hydrolases

Cholinesterase

Acetylcholinesterase Butyrylcholinesterase

Pectinesterase 6-phosphogluconolactonase PAF acetylhydrolase

Lipase

Bile salt-dependent Gastric/Lingual Pancreatic Lysosomal Hormone-sensitive Endothelial Hepatic Lipoprotein Monoacylglycerol Diacylglycerol

Phospholipase

A1 A2 B

3.1.2: Thioesterase

Palmitoyl protein thioesterase Ubiquitin carboxy-terminal hydrolase L1 4-hydroxybenzoyl-CoA thioesterase

3.1.3: Phosphatase

Alkaline phosphatase

ALPI ALPL ALPP

Acid phosphatase
Acid phosphatase
(Prostatic)/Tartrate-resistant acid phosphatase/Purple acid phosphatases Nucleotidase Glucose 6-phosphatase Fructose 1,6-bisphosphatase

Calcineurin

Protein phosphatase

PP2A

OCRL Pyruvate dehydrogenase phosphatase Fructose 6-P,2-kinase:fructose 2,6-bisphosphatase PTEN Phytase Inositol-phosphate phosphatase

IMPA1, IMPA2, IMPA3

Protein phosphatase: Protein tyrosine phosphatase Protein serine/threonine phosphatase Dual-specificity phosphatase

3.1.4: Phosphodiesterase

Autotaxin Phospholipase

C D

Sphingomyelin phosphodiesterase

1

PDE1 PDE2 PDE3 PDE4A/PDE4B PDE5 Lecithinase (Clostridium perfringens alpha toxin) Cyclic nucleotide phosphodiesterase

3.1.6: Sulfatase

arylsulfatase

Arylsulfatase A Arylsulfatase B Arylsulfatase E Steroid sulfatase

Galactosamine-6 sulfatase Iduronate-2-sulfatase N-acetylglucosamine-6-sulfatase

Nuclease
Nuclease
(includes deoxyribonuclease and ribonuclease)

3.1.11-16: Exonuclease

Exodeoxyribonuclease

RecBCD

Exoribonuclease

Oligonucleotidase

3.1.21-31: Endonuclease

Endodeoxyribonuclease

Deoxyribonuclease
Deoxyribonuclease
I Deoxyribonuclease
Deoxyribonuclease
II Deoxyribonuclease
Deoxyribonuclease
IV Restriction enzyme UvrABC endonuclease

Endoribonuclease

RNase III RNase H

1 2A 2B 2C

RNase P RNase A

1 2 3 4/5

RNase T1 RNA-induced silencing complex

either deoxy- or ribo-

Aspergillus nuclease S1 Micrococcal nuclease

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Enzymes

Activity

Active site Binding site Catalytic triad Oxyanion hole Enzyme
Enzyme
promiscuity Catalytically perfect enzyme Coenzyme Cofactor Enzyme
Enzyme
catalysis

Regulation

Allosteric regulation Cooperativity Enzyme
Enzyme
inhibitor

Classification

EC number Enzyme
Enzyme
superfamily Enzyme
Enzyme
family List of enzymes

Kinetics

Enzyme
Enzyme
kinetics Eadie–Hofstee diagram Hanes–Woolf plot Lineweaver–Burk plot Michaelis–Menten kinetics

Types

EC1 Oxidoreductases (list) EC2 Transferases (list) EC3 Hydrolases (list) EC4 Lyases (list) EC5 Isomerases (list) EC6 Ligases (list)

Molecular and Cellular Bi

.