The presence of zinc in the restriction enzyme Eco RI

We have determined that the restriction endonuclease Eco RI contains 1.0 +/- 0.1 eq of zinc/monomeric enzyme. DNA cleavage by Eco RI is inhibited by ortho-phenanthroline after preincubation of the enzyme with the chelating agent. A similar inhibition by the nonchelating meta-phenanthroline is not seen. The sensitivity of the inhibition by the neutral ligand ortho-phenanthroline to preincubation is consistent with the tightly bound and inaccessible nature of the metal site. Extensive dialysis against the ortho-phenanthroline inhibitor leads to the release of the bound metal with the concomitant loss of enzyme activity. The tightly bound Zn2+ cation, then, appears to be necessary for enzyme function. The finding of zinc in Eco RI further illustrates the ubiquity of Zn2+ to DNA-protein complexes.