Document Detail


Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions.
MedLine Citation:
PMID:  7827059     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence, but in the absence of divalent metal ions, it binds DNA without any specificity: gel-shift experiments had revealed multiple EcoRV-DNA complexes, due to the binding of one, two, three, or more molecules of protein per molecule of DNA, with the same equilibrium constant for each association. In this study, the binding of EcoRV to DNA was measured by gel shift in the presence of Ca2+, an ion that perturbs the Mg(2+)-dependent activity of EcoRV but that fails to support DNA cleavage. With Ca2+, and at a lower concentration of EcoRV protein than that required for binding in the absence of divalent metal ions, a single complex was observed with DNA containing the EcoRV recognition site. This complex was not formed with DNA that had been methylated at the EcoRV site nor with an isogenic DNA lacking the EcoRV recognition site. The single complex thus is due to the specific binding of EcoRV to its recognition site on the DNA. From gel shifts with a permuted set of DNA fragments, the degree of DNA bending by EcoRV at its recognition site was estimated to be 53 degrees +/- 4 degrees. This angle is similar to that seen in the crystal structure of the cognate DNA-protein complex. Calcium ions thus appear to mimic the role of Mg2+ in generating a specific protein-metal-DNA complex, but in contrast to Mg2+, Ca2+ gives a stable ternary complex in which the DNA-bound nuclease cannot cleave the DNA.
Authors:
I B Vipond; S E Halford
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  34     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1995 Jan 
Date Detail:
Created Date:  1995-02-23     Completed Date:  1995-02-23     Revised Date:  2009-09-29    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1113-9     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, University of Bristol, U.K.
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MeSH Terms
Descriptor/Qualifier:
Calcium / metabolism*
DNA / metabolism
DNA-Binding Proteins / metabolism*
Deoxyribonucleases, Type II Site-Specific / metabolism*
Escherichia coli / enzymology
Manganese / metabolism*
Methylation
Nucleic Acid Conformation
Substrate Specificity
Grant Support
ID/Acronym/Agency:
//Wellcome Trust
Chemical
Reg. No./Substance:
0/DNA-Binding Proteins; 7439-96-5/Manganese; 7440-70-2/Calcium; 9007-49-2/DNA; EC 3.1.21.4/Deoxyribonucleases, Type II Site-Specific; EC 3.1.21.4/GATATC-specific type II deoxyribonucleases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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