Oligodeoxynucleotides containing 4-thiothymidine and 6-thiodeoxyguanosine as affinity labels for the Eco RV restriction endonuclease and modification methylase.

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RESUMO

4-Thiothymidine and 6-thiodeoxyguanosine were incorporated into synthetic dodecamers containing the recognition site d(GATATC) of the enzymes Eco RV endonuclease and Eco RV methyltransferase. Upon irradiation with long wavelength UV light (340-360 nm), these oligodeoxynucleotides were photochemically crosslinked to both enzymes. The yields were up to 35% with the methyltransferase, but lower (up to 6%) with the endonuclease. Oligodeoxynucleotides containing 4-thiothymidine generally gave higher yields of crosslinking than those containing 6-thiodeoxyguanosine. Although both specific (i.e. those containing the d(GATATC) sequence) and non-specific (lacking this sequence) photoreactive oligodeoxynucleotides gave rise to crosslinked products, the use of a non-reactive, competitive substrate oligodeoxynucleotide suppressed the crosslinking, indicating that the reaction takes place at the enzymes' active sites. Oligodeoxynucleotides containing 4-thiocyanatothymidine or 6-thiocyanatodeoxyguanosine were also prepared by treatment of the title oligomers with CNBr and KCN. The dodecamers containing 4-thiocyanatothymidine were found to covalently modify both enzymes under study, with levels of crosslinking reaching up to 42% with the endonuclease and up to 12% with the methyltransferase. No crosslinking was observed with oligodeoxynucleotides containing 6-thiocyanatodeoxyguanosine.

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