Analysis of the recognition mechanism involved in the EcoRV catalyzed cleavage of DNA using modified oligodeoxynucleotides.
We have prepared a series of undecadeoxynucleotides that contain changes in the functional group pattern present within the EcoRV recognition site - GATATC-. Oligonucleotides were synthesized on solid phase using normal and modified beta-cyanoethylphosphoramidites and analyzed in steady state cleavage experiments with the EcoRV restriction endonuclease. The following groups appear to interact strongly with the enzyme, since their modification or substitution renders the oligonucleotides refractory to cleavage: the exocyclic NH2-groups of both A residues, the N7 of the first A residue, the exocyclic NH2-group of the C residue and the CH3-groups of both T residues. The exocyclic NH-group of the G residue supports effective recognition, since its absence lowers the kcat of the cleavage reaction. The N7 of the second A residue and the C5 position of the C residue apparently are not recognized by EcoRV; their substitution by -CH- or modification with -Br or -CH3, resp., does not considerably change the rate of cleavage. All oligonucleotides investigated compete with the unmodified substrate for binding to the enzyme. We conclude that EcoRV recognizes its substrate presumably through hydrogen bonds to the exocyclic NH2-group and the N7 of the first A residue, the exocyclic NH2-groups of the second A and the C residue, as well as through hydrophobic interactions with both T residues.
Fliess
A
A
Zentrum Biochemie, Medizinische Hochschule Hannover, FRG.
Wolfes
H
H
Seela
F
F
Pingoud
A
A
D000225:Adenine; D001483:Base Sequence; D002384:Catalysis; D003596:Cytosine; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000378:metabolism; D013941:Thymine
D015345:Oligonucleotide Probes; D006147:Guanine; D003596:Cytosine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D000225:Adenine; D013941:Thymine