Oligodeoxynucleotides containing 4-thiothymidine and 6-thiodeoxyguanosine as affinity labels for the Eco RV restriction endonuclease and modification methylase.
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.
Nikiforov
T T
TT
Department of Biochemistry, University of Southampton, Bassett Crescent East, UK.
Connolly
B A
BA
D000345:Affinity Labels / Q000737:chemistry*; D001483:Base Sequence; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013872:Thionucleosides / Q000737:chemistry*; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry
D000345:Affinity Labels; D009838:Oligodeoxyribonucleotides; D013872:Thionucleosides; D006151:Guanosine; C070456:4'-thiothymidine; D004247:DNA; C005783:6-thioguanosine; C056127:DNA modification methylase EcoRV; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D013936:Thymidine