EcoRI is an endonuclease

Endonuclease

Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...

 enzyme isolated from strains of E. coli, and is part of the restriction modification system

Restriction modification system

The restriction modification system is used by bacteria, and perhaps other prokaryotic organisms to protect themselves from foreign DNA, such as the one borne by bacteriophages. This phenomenon was first noticed in the 1950s. Certain bacteria strains were found to inhibit the growth of viruses...

.

In molecular biology it is used as a restriction enzyme

Restriction enzyme

A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...

. It creates sticky ends

DNA end

DNA end or sticky end refers to the properties of the end of a molecule of DNA or a recombinant DNA molecule. The concept is important in molecular biology, especially in cloning or when subcloning inserts DNA into vector DNA. All the terms can also be used in reference to RNA. The sticky ends or...

 with 5' end overhangs. The nucleic acid sequence where the enzyme cuts is GAATTC, which, as the complementary sequence is CTTAAG, has rotational symmetry

Rotational symmetry

Generally speaking, an object with rotational symmetry is an object that looks the same after a certain amount of rotation. An object may have more than one rotational symmetry; for instance, if reflections or turning it over are not counted, the triskelion appearing on the Isle of Man's flag has...

.

Primary Structure

EcoRI contains the PD..D/EXK motif within its active site like many restriction endonucleases

Restriction enzyme

A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...

. It is typically used in the isolation and restriction of bacterial plasmid DNA. In EcoRI this motif consists of residues P90, D91, E111, A112, K113(2)1.

Tertiary and Quaternary Structure

The enzyme is a homodimer of a 31 kilodalton subunit consisting of one globular domain of the α/β architecture. Each subunit contains a loop which sticks out from the globular 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 http://www.pdb.org/pdb/explore.do?structureId=1QPS of the complex. The solved crystal structure shows that the subunits of the enzyme homodimer interact with the DNA symmetrically(3). In the complex, two α-helices

Alpha helix

A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...

 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 that is believed to allow the enzyme's two active sites to communicate(4).

Uses

Restriction enzymes such as EcoRI are used in a wide variety of molecular genetics techniques including cloning, DNA screening and deleting sections of DNA in vitro. Restriction enzymes like EcoRI that generate sticky ends of DNA are often used to cut DNA prior to ligation, as the sticky ends make the ligation reaction more efficient. EcoRI can exhibit non site-specific cutting, known as star activity

Star activity

Star activity is a relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimum for the enzyme...

, depending on the conditions present in the reaction. Conditions that can induce star activity when using EcoRI include low salt concentration, high glycerol concentration, excessive amounts of enzyme present in the reaction, high pH and contamination with certain organic solvents.

See also

• EcoRII, another nuclease enzyme from E. coli.
• EcoRV
  EcoRV
  EcoRV 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'-GAT|ATC-3' and makes...
  
  , another nuclease enzyme from E. coli.