EcoRI
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EcoRI (pronounced "eco R one") is a nuclease enzyme isolated from certain strains of E. coli, and is part of the restriction modification system.
In molecular biology, it is a commonly used restriction enzyme. It creates sticky ends with 5' end overhangs. The nucleic acid sequence where the enzyme cuts is G|AATTC, which is a palindrome as the complementary sequence is CTTAA|G.
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[edit] Structure
[edit] Primary Structure
EcoRI contains the PD..D/EXK motif within its active site like many restriction endonucleases. In EcoRI this motif consists of residues P90, D91, E111, A112, K113(2).
[edit] Tertiary and Quaternary Structure
The enzyme is made up of one globular domain of the α/β architecture. It also contains a loop which sticks out from the single domain and wraps around the DNA when bound (3).
EcoRI has been cocrystallized with the sequence it normally cuts. This crystal was used to solve the structure [[1]] of the complex. The solved crystal structure shows the the enzyme forms a homodimer when it binds DNA where both subunits interact with the DNA symmetrically(3). In the complex, two α-helices from each subunit come together to form a four helix bundle(2). On the interacting helices are residues Glu144 and Arg145 which interact together forming a crosstalk ring allowing the active sites to communicate(4).
[edit] Uses
Because of their ability to cut DNA in predictable locations and leave ends which can be ligated back together, they are commonly used in cloning, DNA screening, deletion mutagenesis, and many other commonly used techniques. EcoRI is prone to exhibit star activity if an excess of salts are present during the digestion.
[edit] See also
- EcoRV, another nuclease enzyme from 'E. coli.
- FokI, another nuclease enzyme from Flavobacterium okeanokoites
[edit] References
1. Bitinaite, J., D. A. Wah, Aggarwal, A. K., Schildkraut, I. (1998). "FokI dimerization is required for DNA cleavage." Proc Natl Acad Sci U S A 95(18): 10570-5.
2. Pingoud, A., Jeltsch, A. (2001) Structure and function of type II restriction endonucleases, Nucl. Acids Res. 29. 18, 3705-3727.
3. McClarin, J.A., Frederick, C.A., Wang, B.C., Greene, P., Boyer, H.W., Grable, J., Rosenberg, J.M. (1986) Structure of the DNA-EcoRI endonucleases recognition complex at 3 Å resolution, Science. 234. 4783, 1526-1542.
4. Kurpiewski, M.R., Engler, L.E., Wozniak, L.A., Kobylanska, A., Koziolkiewicz, M., Stec, W.J, Jen-Jacobson, L. (2004) Mechanisms of coupling between DNA recognition and catalysis in EcoRI endonucleases, Structure 12. 1775-1788.
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