The activity of the EcoRV restriction endonuclease is influenced by flanking DNA sequences both inside and outside the DNA-protein complex

Abstract

The EcoRV restriction endonuclease cleaves DNA not only at its recognition sequence but also at most other sequences that differ from the recognition site by one base pair. Compared to the reaction at the recognition site, the reactions at noncognate sites are slow but 1 out of the 12 noncognate sites on the plasmid pAT153 is cleaved more than 50 times faster than any other. The increase in the reaction rate at the preferred noncognate site, relative to other sites, was caused by the DNA sequences in the 4 base pairs from either side of the site. For enhanced activity by EcoRV, particular bases were needed immediately adjacent to the site, inside the DNA-protein complex. At these loci, the protein interacts with the phosphate groups in the DNA and the flanking sequence may control the activity of the enzyme by determining the conformation of the DNA, thus aligning the phosphate contacts. But the preferential cleavage also depended on sequences further away from the site, at loci outside the complex. At external positions, beyond the reach of the protein, the EcoRV enzyme required flanking sequences that give rise to flexibility in DNA conformation. These may facilitate the distortion of the DNA required for catalysis by EcoRV.