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Restriction Endonuclease EcoRI Alters the Enantiomeric Preference of Chiral Metallointercalators for DNA: An Illustration of a Protein-Induced DNA Conformational Change

Barton, Jacqueline K. and Paranawithana, Shanthi R. (1986) Restriction Endonuclease EcoRI Alters the Enantiomeric Preference of Chiral Metallointercalators for DNA: An Illustration of a Protein-Induced DNA Conformational Change. Biochemistry, 25 (8). pp. 2205-2211. ISSN 0006-2960. http://resolver.caltech.edu/CaltechAUTHORS:20160516-132218229

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Abstract

A conformational change in the DNA plasmid ColE_1 appears to occur upon specific binding of the restriction endonuclease EcoRI. Enzyme association alters the chiral discrimination found in binding metallointercalators to DNA sites. The complexes tris(l,10-henanthroline)ruthenium(II), Ru(phen)_3^(2+), tris( 4,7-diphenyl-1, 10-phenanthroline) ruthenium(II), Ru(DIP)_3^(2+), and tris( 4,7-diphenyl- l, 10-phenanthroline)cobalt(III), Co(DIP)_3^(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the Δ configuration bound preferentially by right-handed B-DNA. In the presence of EcoRI, however, this enantioselectivity is altered. The chiral intercalators, at micromolar concentrations, inhibit the reaction of EcoRI, but for each enantiomeric pair it is the Λ enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially. Kinetic studies in the presence of Λ-Ru(DIP)_3^(2+) indicate that the enzyme inhibition occurs as a result of the Λ enantiomer binding to the enzyme-DNA complex as well as to the free enzyme. Furthermore, photolytic strand cleavage experiments using Co(DIP)_3^(3+) indicate that the metal complex interacts directly at the protein-bound DNA site. Increasing concentrations of bound EcoRI stimulate photoactivated cleavage of the DNA helix by Λ-Co(DIP)_3^(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme. Thus, although Λ-Co(DIP)_3^(3+) does not bind closely to the DNA in the absence of enzyme, specific binding of EcoRI appears to alter the DNA structure so as to permit the close association of the Λ isomer to the DNA helix. Mapping experiments demonstrate that this association leads to photocleavage of DNA by the cobalt complex at or very close to the EcoRI recognition site. This study provides evidence that in solution, under enzymatic conditions, a DNA-binding protein may distort the DNA helical structure and furthet illustrates how small molecular probes of DNA conformation might be used in examining the structure of protein-bound DNA sites.


Item Type:Article
Related URLs:
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http://dx.doi.org/10.1021/bi00356a053DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi00356a053PublisherArticle
ORCID:
AuthorORCID
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 1986 American Chemical Society. Received March 7, 1985; Revised Manuscript Received November 8, 1985. J.K.B. is a Research Fellow of the Alfred P. Sloan Foundation and an NSF Presidential Young Investigator. This work was also generously supported by Grant GM 32203 from the National Institutes of Health.
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Funding AgencyGrant Number
Alfred P. Sloan FoundationUNSPECIFIED
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NIHGM-32203
Record Number:CaltechAUTHORS:20160516-132218229
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160516-132218229
Official Citation:Restriction endonuclease EcoRI alters the enantiomeric preference of chiral metallointercalators for DNA: an illustration of a protein-induced DNA conformational change Jacqueline K. Barton and Shanthi R. Paranawithana Biochemistry 1986 25 (8), 2205-2211 DOI: 10.1021/bi00356a053
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67133
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:16 May 2016 21:45
Last Modified:16 May 2016 21:45

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