The presence of zinc in the restriction enzyme Eco RI.

J K Barton,L A Basile,S R Paranawithana

doi:10.1016/s0021-9258(18)34267-4

Abstract

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.

Highlights

Zinc is essential to nucleic acid processing (1, 2 )
In an effort to extend the characterizaotifoDnNA enzymes by their metal content and to find a system which would facilitate chemical studies of the metal center, we have examined Eco RI, a type I1 restriction enzyme from Escherichia coli [17,18,19]
The type I1 restriction enzymes are among the simplest DNA sequence-specific enzymes known [19,20,21]

Summary

The Presence of Zinc in the Restriction Enzyme ECORI*

Isolated [23,24].We report here thaEtco RI contains a tightly bound zinc ion. The zinc ion appears necessary to enzyme function. We have determined that the restriction endonuclease Eco RI contains 1.0 f 0.1 eq of zinc/monomeric enzyme. DNA c!eavage by Eco RI is inhibited by orthophenanthroline after preincubation of the enzyme with the chelating agent. 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. The tightly bound Zn2+cation, appears to be necessary for enzyme function.The finding of zinc in Eco RI furtherillustrates the ubiquity of Zn2+to DNA-protein complexes. The zinc dication appears as an integral part of enzymes which bind polynucleotides either as substrate or template.

RI binds to theduplex palindromic sequence

American University

EXPERIMENTAL PROCEDURES

Zinc content of Eco RI

AND DISCUSSION