INFLUENCE OF THE PHOSPHATE BACKBONE ON THE RECOGNITION AND HYDROLYSISOF DNA BY THE ECORV RESTRICTION-ENDONUCLEASE - A STUDY USING OLIGODEOXYNUCLEOTIDE PHOSPHOROTHIOATES

A set of phosphorothioate-containing oligonucleotides based on pGACGAT ATCGTC, a self-complementary dodecamer that contains the EcoRV recogni tion sequence (GATATC), has been prepared. The phosphorothioate group has been individually introduced at the central nine phosphate positio ns and the two diastereomers produced at each site separated and purif ied. The K-m and V-max values found for each of these modified DNA mol ecules with the EcoRV restriction endonuclease have been determined an d compared with those seen for the unmodified all-phosphate-containing dodecamer, This has enabled an evaluation of the roles that both of t he non-esterified oxygen atoms in the individual phosphates play in DN A binding and hydrolysis by the endonuclease, The results have also be en compared with crystal structures of the EcoRV endonuclease, complex ed with an oligodeoxynucleotide, to allow further definition of phosph ate group function during substrate binding and turnover, For further study, see the related article ''Probing the Indirect Readout of the R estriction Enzyme EcoRV: Mutational Analysis of Contacts to the DNA Ba ckbone'' (Went, A., Jeltsch, A, and Pingoud, A, (1996) J. Biol, Chem, 271, 5565-5573).