ECORV-T94V - A MUTANT RESTRICTION-ENDONUCLEASE WITH AN ALTERED SUBSTRATE-SPECIFICITY TOWARDS MODIFIED OLIGODEOXYNUCLEOTIDES

Synthetic oligodeoxynucleotides with single methyl phosphonate (mp) su bstitutions were used for an analysis of the contribution of phosphate contacts to the recognition of the cleavage site by the restriction e ndonuclease EcoRV. Only in the last position within the recognition se quence, is the methyl phosphonate substitution tolerated by the enzyme . The wild-type enzyme cleaves the Sr diastereomer of the oligodeoxynu cleotide GACGATAT(mp)CGTC and the unmodified sequence with equal rates , whereas the Rp diastereomer is cleaved much more slowly Inspection o f the crystal structure of an EcoRV-DNA complex revealed that the non- bridging oxygen atoms of the phosphodiester bond between the T and C b ases are in hydrogen bonding distance of the hydroxyl group of the ami no acid Thr94. We therefore tried to engineer a variant of EcoRV that would prefer a methyl phosphonate linkage over a normal phosphodiester bond and produced mutants with amino acid exchanges at position 94. O ne of them, Thr94Val, shows a dramatically reduced activity towards th e unmodified DNA and does not accept the Rp diastereomer, but cleaves the Sp diastereomer with the same rate as wildtype EcoRV. Its selectiv ity, i.e. the ratio of cleavage rates determined for the unmodified an d modified substrates, differs by three orders of magnitude from that of the wildtype enzyme.