INTERACTION OF THE RESTRICTION-ENDONUCLEASE ECORV WITH THE DEOXYGUANOSINE AND DEOXYCYTIDINE BASES IN ITS RECOGNITION SEQUENCE

The interaction of the EcoRV restriction endonuclease with the dG and dC bases in its recognition sequence (GATATC) has been studied using b ase analogues. These modified dG and dC bases each have a single poten tial protein contact removed. The analogues have been incorporated int o the self-complementary dodecamer d(pGACGATATCGTC) at the appropriate positions (underlined). Many of the analogues caused no change in the T-m of the duplex or else lowered the T-m by a small amount such that a duplex was still formed at temperatures suitable for enzyme assay. However, the dG analogue 2-aminopurine-1-beta-D-2'-deoxyriboside desta bilized the duplex to such an extent that the 12'-mer could not be use d for enzyme assays. To overcome this, a longer self-complementary 18' -mer was used with this modified base. The circular dichroism spectra of the modified base containing 12'-mers (and the 18'-mer in the case of 2-aminopurine) were very similar to the parent sequences lacking mo dified bases. This demonstrates the formation of B-DNA structures in a ll cases and similar overall conformations. The K-m and k(cat) values for the various modified oligomers have been determined, and these dat a have been used to assess the roles that functional groups on the dG and dC bases play in the recognition and hydrolysis of GATATC sequence s by the endonuclease. The results obtained here have been compared to the crystal structures of the EcoRV complexed with a GATATC sequence, and this has allowed a critical evaluation of the base analogue appro ach. Both methods indicate that the 6-keto oxygen and 7-ring nitrogen of dG exposed in the major groove are vital for DNA recognition and hy drolysis.