Sandbox 666: Difference between revisions

Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.

   

''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians.  In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.

    

''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg²⁺. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.

== Structure ==

| [[Image:Image1 EcoRI.png |thumb| The topology of EcoRI restriction endonuclease<ref name="A" />.× catalytically important amino acid residues, • amino acid residues in interactions with DNA, pink regions: dimerization contacts|right|385px]]

|}

==Catalytic mechanism<ref name="A" />==

The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*10⁶ bp.s^-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten.  This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.

It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA.  

During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg²⁺, play also an essential role in the recognition process. The two homodimeres of ''Eco''RI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved.  

Specific binding bend the DNA by about 12° and involve interactions between ''Eco''RI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between ''Eco''RI and the recognition sequence is maximal.  

The restriction enzymes are highly specific and highly cooperative.

== Application of EcoRI in molecular biology ==

    

Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.

== Links ==

[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)

== References ==

<references/>

== Contributor ==

Raphaël BILGER,  Virginie GROSBOILLOT