SUBSTRATE-ASSISTED CATALYSIS IN THE CLEAVAGE OF DNA BY THE ECORI AND ECORV RESTRICTION ENZYMES

The crystal structure analyses of the EcoRI-DNA and EcoRV-DNA complexe s do not provide clear suggestions as to which amino acid residues are responsible for the activation of water to carry out the DNA cleavage . Based on molecular modeling, we have proposed recently that the atta cking water molecule is activated by the negatively charged pro-R(P) p hosphoryl oxygen of the phosphate group 3' to the scissile phosphodies ter bond. We now present experimental evidence to support this proposa l. (i) Oligodeoxynucleotide substrates lacking this phosphate group in one strand are cleaved only in the other strand. (ii) Oligodeoxynucle otide substrates carrying an H-phosphonate substitution at this positi on in both strands and, therefore, lacking a negatively charged oxygen at this position are cleaved at least four orders of magnitude more s lowly than the unmodified substrate. These results are supported by ot her modification studies: oligodeoxynucleotide substrates with a phosp horothioate substitution at this position in both strands are cleaved only if the negatively charged sulfur is in the R(P) configuration as shown for EcoRI [Koziolkiewicz, M. & Stec, W. J. (1992) Biochemistry 3 1, 9460-9466] and EcoRV (B. A. Connolly, personal communication). As t he phosphate residue 3' to the scissile phosphodiester bond is not nee ded for strong DNA binding by both enzymes, these findings strongly su ggest that this phosphate group plays an active role during catalysis. This proposal, furthermore, gives a straightforward explanation of wh y in the EcoRI-DNA and EcoRV-DNA complexes the DNA is distorted differ ently, but in each case the 3' phosphate group closely approaches the phosphate group that is attacked. Finally, an alternative mechanism fo r DNA cleavage involving two metal ions is unlikely in the fight of ou r finding that both EcoRI and EcoRV need only one Mg2+ per active site for cleavage.