Nutrition Guide

TaqI is a restriction enzyme isolated from the bacterium Thermus aquaticus in 1978.[1] It has a recognition sequence of

Structure-based sequence alignment and site-directed mutagenesis identified the putative PD..D/EXK active sites of the EcoRII catalytic domain dimer that in apo structure are spatially blocked by the N-terminal domains.[6]

The C-terminal catalytic domain has a typical[10] restriction endonuclease-like fold (SCOP 52979) and belongs to the large (more than 30 members) restriction endonuclease superfamily (SCOP 52980).

The apo crystal structure of EcoRII mutant R88A (PDB 1NA6)[6] has been solved at 2.1 Å resolution. The EcoRII monomer has two domains, N-terminal and C-terminal, linked through a hinge loop.

NdeI is an endonuclease isolated from Neisseria denitrificans.

In molecular biology, it is commonly used as a restriction enzyme.

Recognition sequence of NdeI:

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.

Like EcoRI, 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 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 is the only type II restriction endonuclease known to cause a major protein-induced conformational change in the DNA. [

EcoRV has been cocrystallized with the sequence it normally cuts. This crystal was used to solve the structure [1] of the complex.

The core of the enzyme consists of a five-stranded mixed ?-sheet flanked by ?-helices. The core is conserved in all other type II restriction endonucleases. It also has an N-terminal dimerization subdomain formed by a short ?-helix, a two-stranded antiparallel -sheet, and a long ?-helix. This subdomain is found only in EcoRV and PvuII. [1]

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′-GAT|ATC-3′ and makes a cut at the vertical line. The complementary sequence is then 3′-CTA|TAG-5′. The ends are blunt and can be ligated into a blunt cloning site easily but with lower efficiency than sticky ends.

HindIII as well as other type II restriction endonucleases are very useful in modern science, particularly in DNA sequencing and mapping. Unlike type I restriction enzymes, type II restriction endonucleases perform very specific cleaving of DNA. Type I restriction enzymes recognize specific sequences, but cleave DNA randomly at sites other than their recognition site whereas type II restriction enzymes cleave only at their specific recognition site.[7] Since their discovery in the early 1970s, type II restriction enzymes have revolutionized the way scientists work with DNA, particularly in genetic engineering and molecular biology.

Major uses of type II restriction enzymes include gene analysis and cloning. They have proven to be ideal modeling systems for the study of protein-nucleic acid interactions, structure-function relationships, and the mechanism of evolution.[2] They make good assays for the study of genetic mutations by their ability to specifically cleave DNA to allow the removal or insertion of DNA. Through the use of restriction enzymes, scientists are able to modify, insert, or remove specific genes, a very powerful tool especially when it comes to modifying an organism’s genome.