Restriction enzyme EcoRV/DNA complex (before DNA cleave, without Mg2+ ion)
Escherichia coli (bacteria)
The restriction enzymes play important roles in the self-defense mechanism in which the enzyme cleaves and excludes various viruses or exogenous DNAs invading the cell. These enzymes protect their own DNAs by methylating cytosines and adenines located on their specific recognition sequences. These enzymes are endonucleases, which cleave inner 3'5'-phosphodiester bonds on polynucleotide chains, and are classified into the types I or II. The type I enzymes recognize the specific sequences but don't cleave specifically. Type II enzymes cleave double-strand DNAs (dsDNA) at specific sites. EcoRV derived from Escherichia coli is one of the best studied type II restriction endonucleases, both functionally and structurally. This enzyme is active in the presence of Mg2+, and cleaves dsDNA at the recognition DNA sequence at least 106 times faster than at any other DNA sequence. On the other hand in the absence of Mg2+, EcoRV doesn't recognize the specific sequences.
The three crystal structures of EcoRV-substrate complexes without Mg2+ and with Mg2+, and EcoRV-product complex with Mg2+ shown that the specificity of EcoRV is caused by the binding of Mg2+ and the conformational changes induced by Mg2+ binding. The DNA recognition sites named R-loops in EcoRV tightly bind in the major grooves of dsDNA by hydrogen bond interactions, and induce a kink in the DNA. When Mg2+ comes near the enzyme, acidic residues, e.g. Asp, and negatively charged phosphate groups on the recognition sequence of DNA approach each other, and form the Mg2+ binding site. By Mg2+ binding at this site, kinked DNA is stabilized. Subsequently EcoRV cleaves dsDNA. After the cleavage, keeping the kink introduced into the DNA, the phosphate group of the cleaved DNA fragment is pulled to Mg2+ and the base groups produce the stacking interactions between adjacent strands to stabilize them. Thus it is shown that the Mg2+ binding is essential for the specific recognition and cleavage of dsDNA by EcoRV.
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: Yuko Tsuchiya