Structure-function correlation for the EcoRV restriction enzyme: from non-specific binding to specific DNA cleavage.
The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg2+; but in binding to DNA in the absence of Mg2+, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg2+ to EcoRV-DNA complexes: the complex at the recognition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of the EcoRV endonuclease, and of its complexes with either specific or non-specific DNA, have been solved by X-ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non-specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg2+. Without this distortion, the EcoRV-DNA complex would be unable to bind Mg2+ and thus unable to cleave DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Vipond
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IB
Department of Biochemistry, University of Bristol, UK.
Halford
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SE
D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D008274:Magnesium