Eco-RI endonuclease (Eco-RI) is a globular type II restriction enzyme found in the bacteria Escherichia coli. This endonuclease functions as a defense mechanism, like many others in bacteria and archaea, to protect the organism from invading foreign DNA. The bacteria’s own DNA will remain unaffected due to protective methylation at the specific target site nucleotides, which is put into place immediately after replication (9). As a type II restriction enzyme, Eco-RI binds and cleaves double stranded DNA at specific base pairs. It achieves this cleavage by catalyzing the hydrolysis of the target DNA’s phosphodiester bonds (3). The specificity of these enzymes are usually contained to four to six base pairs and needs magnesium cations as cofactors for catalysis (9). 

Generally speaking, Eco-RI catalyzes cleavage of the DNA sequence GAATTC between the guanine and adenine leaving sticky DNA hanging ends. It accomplishes this by forming a homodimeric structure that binds each DNA strand inducing a kink in DNA (1). Furthermore, in characterizing the crystallized protein, it was found that activity depended on the amount of magnesium ions in solution suggesting that the magnesium ions trigger an assemblage in the active site (1). 

The primary structure of Eco-RI consists of 277 amino acids with a molecular weight of 30928.1 Daltons. Given that the average amino acid has a molecular mass of 100 Daltons, the molecular weight is very close to but slightly above the average weight that would be expected. This suggests that most amino acids are generally average with a portion on the larger side. The isoelectric point, or pH point at which the molecule would have no net charge, is 7.77 (7).

The secondary structure of Eco-RI consists of alpha helices, beta sheets, and 3/10 helices to form the globular protein. The alpha helices are mainly on the outside of the protein, while the 3/10 helices make up a lot of the interior. The beta sheets consist of both antiparallel and parallel motifs that help foundationally aid in strand scission and sequence-recognition, which occurs in the helices (1). Overall Eco-RI is about 20% beta sheets, 10% 3/10 helices and more than 50% alpha helices (7).

The tertiary structure of Eco-RI includes only an alpha subunit that binds to one strand of DNA. The polar and nonpolar residues are arranged with mostly nonpolar amino acids on the interior and polar amino acids on the exterior. This maximizes hydrophobic effect and van der waals interactions by allowing external polar molecules to interact with the water based environment of the cell rather than water molecules losing entropy by forming regimented structures around nonpolar external residues (3). An exception in this protein to a mostly nonpolar interior is seen most prominently in the 3/10 helices, which are made of solely basic and polar amino acids in the interior. This charged interior is due to the designated substrate of this enzyme, namely a negatively charged phosphodiester backbone of DNA (9).

The quaternary structure of Eco-RI is a homodimer that is made up of many motifs, four of which are of note. The extended chain motif extends from Met-137 to Ala-142 and runs through the major groove of the DNA, parallel to its backbone. This site is a mutational hotspot that makes contacts with mostly pyrimidine bases. The beta bridge connects between β1 and β2 to extend over the gap between globular units and is made up of an alpha helix as well as a pair of antiparallel beta strands. This bridge forms one part of the structure where the DNA backbone is held. The placement of Glu-111 is of particular importance as mutations here can reduce the cleavage activity of the enzyme without altering the DNA binding. Lastly, a beta-loop-alpha motif is seen connecting β3 to α4 and β4 to α5 forming arms that surround the DNA and contact the backbone. This last motif plays a general topological role in nucleic acid binding (2).

From these structures, derives the function of the Eco-RI enzyme. Many important amino acids in particular work specifically to aid in the mechanism of cleaving DNA (3). Most notably, thebinding site of the Eco-RI enzyme binds the DNA ligand. Lys-89 and Lys-113 allow this specificity to occur by hydrogen bonding to the nucleotides flanking the guanine-adenine cleavage point. This results in electron withdraw that will eventually allow the phosphodiester bond to be cleaved with staggered or sticky ends (9).  

Prior to this, Asp-91 and Glu-111 work to bind to the cofactor magnesium cations. This serves to help bind to and position DNA as well as partially neutralize negative charges from phosphorous in the transition state. Additionally at this site, the water molecules are polarized toward deprotonation to attack the phosphoryl groups of the DNA ultimately leaving a free 3’-hydroxyl group and 5’-phosphoryl group product (9). 

In order to physically cleave the DNA double strand, Eco-RI utilizes an inversion of the phosphodiester backbone to produce a kink. This occurs due to the malleability of the 5’-TA-3’ sequence with its partner 3’-AT-5’. Asn-141 and Arg-145 have bidentate hydrogen bonds with the DNA substrate at the scissile bond, which result from adjacent hydroxyls groups interacting with a different atom of the same planar polar chain residue. Also involved in the active sites of Eco-RI are the Tyr-192 and Glu-193 residues. These residues are located in the random coil that crosses over to the DNA of the other dimer. Both of these sets of amino acids are thought to be aiding in correct recognition of the DNA sequence GAATTC palindrome with Asn-141 and Arg-145 recognizing the DNA bound to its specific dimer and Tyr-192 and Glu-193 recognizing the DNA of the other dimer. This system, as with most biological systems, has many recognition sites on each of the dimers respectively as well as crossover between the two to act as a failsafe (9).

In order to find homologous protein structures, the PSI- BLAST database and Dali were used. PSI-BLAST showed that Eco-RI has a similar primary structure to the ApoI gene with an E value of 2e-04, meaning that they are indeed very similar proteins. This gene functions also as an endonuclease in Arthrobacter protophormiae (5). Dali showed that Sgr-AI(3DVO) had a similar secondary and tertiary structure to Eco-RI with a z score of 7.6. Although this is a relatively low score, the Sgr-AI gene functions similarily as a type II restriction enzyme by also hydrolyzing DNA. Although accomplishing the same function, Sgr-AI is a larger protein at 338 amino acids and very importantly makes a tetramer complex that includes both manganese and calcium cations. For this reason the structures of Eco-RI and Sgr-AI do not overlap well (6). Sgr-AI also has a different proportion of secondary structures with a lower emphasis on beta sheets and higher emphasis on alpha helices (10). This could be due to the larger size and therefore surface area of the Sgr-AI enzyme. The external alpha helices would need to span a greater area to maximize hydrophobic affect.

The real impact of this gene is seen in its human analogous form. This analog, also called Eco-RI, is correlated to infantile facioscapulohumeral muscular dystrophy. It was found that the length of the Eco-RI fragment in the chromosome 4q35 region affected the severity of the phenotype with shorter Eco-RI fragments having a more severe phenotype. This disease has symptoms in every possible organ of the body due to its derivation in the inherent DNA nuclease function and therefore is incredibly debilitating for the infants having the disease (4).