CHEMICAL CROSS-LINKING OF MVAI AND ECORII ENZYMES TO DNA DUPLEXES CONTAINING MONOSUBSTITUTED PYROPHOSPHATE INTERNUCLEOTIDE BOND

DNA duplexes containing a monosubstituted pyrophosphate internucleotid e group, instead of a phosphodiester bond, were used as cross-linking reagent for the affinity modification of the restriction endonucleases EcoRII and MvaI (R . EcoRII and R . MvaI). An active group was introd uced into the enzyme's recognition site or between the recognition sit e and flanking sequence. The substrate properties of such DNA duplexes were determined. Cross-linking specificity was demonstrated by compet ition experiments with unmodified substrate, as well as by the absence of cross-linking to an active duplex lacking a recognition site. It w as shown that the nucleophilicity of the buffer solution and the prese nce of the enzyme cofactor Mg2+ dramatically affected the cross-linkin g yield.