Chemical cross-linking of MvaI and EcoRII enzymes to DNA duplexes containing a monosubstituted pyrophosphate internucleotide bondстатья
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Дата последнего поиска статьи во внешних источниках: 18 июля 2013 г.
Аннотация:DNA duplexes containing a monosubstituted pyrophosphate internucleotide 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 introduced into the enzyme's recognition site or between the recognition site and flanking sequence. The substrate properties of such DNA duplexes were determined. Cross-linking specificity was demonstrated by competition experiments with unmodified substrate, as well as by the absence of cross-linking to an active duplex lacking a recognition site. It was shown that the nucleophilicity of the buffer solution and the presence of the enzyme cofactor Mg2+ dramatically affected the cross-linking yield.