STRUCTURE-FUNCTION CORRELATION FOR THE ECORV RESTRICTION ENZYME - FROM NONSPECIFIC-BINDING TO SPECIFIC DNA CLEAVAGE

The EcoRV restriction endonuclease cleaves DNA at its recognition sequ ence at least a million times faster than at any other DNA sequence. T he only cofactor it requires for activity is Mg2+; but in binding to D NA in the absence of Mg2+, the EcoRV enzyme shows no specificity for i ts recognition site. Instead, the reason why EcoRV cuts one DNA sequen ce faster than any other is that the rate of cleavage is controlled by the binding of Mg2+ to EcoRV-DNA complexes: the complex at the recogn ition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of the EcoR V endonuclease, and of its complexes with either specific or non-speci fic DNA, have been solved by X-ray crystallography. In the specific co mplex, the protein interacts with the bases in the recognition sequenc e and the DNA takes up a highly distorted structure. In the non-specif ic complex with an unrelated DNA sequence, there are virtually no inte ractions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions bal ances that required for the distortion of the DNA. The distortion inse rts the phosphate at the scissile bond into the active site of the enz yme, where it forms part of the binding site for Mg2+. Without this di stortion, the EcoRV-DNA complex would be unable to bind Mg2+ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.