Restriction enzyme EcoRV / DNA Complex (after DNA cleaved, with Mg2+ ion)
Escherichia coli (bacteria)
Dexoyribonucleases come in two flavors: DNases and endonucleases. DNases cut the dexoyribonucleic acid (DNA) at arbitrary positions, endonucleases, also called restriction enzymes or restriction endonucleases, cut DNA at very specific position. One of the best studied endonucleases, called EcoRV endonuclease, for instance is specific to cut at DNA at the recognition sequence GATATC. It cuts DNA at this position more than a million times faster than it cuts at other positions. For this to be effective, magnesium ions are necessary. In the absence of magnesium the specificity is lost. The restriction enzymes have become very important tools in molecular biology and a good understanding of their activity is therefore of interest. A study was carried out to determine the structure of EcoRV endonuclease together with DNA and magnesium. For the DNA the sequence AAAGATATCTT was chosen, containing the recognition sequence in the middle.
EcoRV endonuclease is a dimer with the two molecules forming a cleft in which the DNA substrate binds. As one can see in this structure the DNA has been cleaved. The DNA does appear buried within the protein in this structure. This is caused by a loop, which folds around the DNA after it has bound. There is one such loop per monomer, called the recognition loop, because it makes contacts with the recognition sequence of the DNA. One can also see that the ends of the cleaved sequence have two magnesium ions bound to their phosphate groups by coordination. These magnesium ions coordinate not only with the DNA phosphate but also with oxygens from the protein. This illustrates the mechanism by which EcoRV endonuclease cleaves DNA.
Protein Data Bank (PDB)
Kostrewa, D. Winkler, F.K.; "Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution."; Biochemistry; (1995) 34:683-696 PubMed:7819264.
author: Arno Paehler