Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases.
We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV.
D001483:Base Sequence; D004247:DNA; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D013936:Thymidine
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D013936:Thymidine