EVIDENCE FOR SUBSTRATE-ASSISTED CATALYSIS IN THE DNA CLEAVAGE OF SEVERAL RESTRICTION ENDONUCLEASES

Substrate-assisted catalysis was suggested to be involved in the DNA c leavage reaction of the restriction endonucleases (ENases) EcoRI and E coRV, because experimental evidence exists that the phosphate group 3' to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cl eavage rates of modified and unmodified oligodeoxyribonucleotides (oli gos), in which the phosphate group 3' to the scissile bond is substitu ted by a methyl phosphonate, were measured for 17 enzymes. Only five t urned out not to be inhibited by this modification (BglII, BstI, BstYI , Cfr10I and MunI); all others cleave the modified substrate at a stro ngly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibit ion of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (BnmHI, Bsp1 43I, Eco72I, MflI, NdeII, Sau3AI, XhoII). The others (AluI, Cfr9I, Dpn II, MboI, PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, clea ve the unmodified strand with a reduced rate, too. Based on these resu lts we conclude that BamHI, Bsp143I, Cfr9I, DpnII, Eco72I, MboI, MflI, NdeII, PvuII, Sau3AI and XhoII may possibly employ substrate assistan ce in catalysis.