Lim, Heon Man and Simon, Melvin I. (1992) The role of negative supercoiling in Hin-mediated site-specific recombination. Journal of Biological Chemistry, 267 (16). pp. 11176-11182. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:LIMjbc92a
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A series of biochemical assays were developed and performed to monitor the molecular events that occur during the Hin-mediated DNA inversion reaction. These events can be divided into five different stages: 1) binding of proteins (Hin, Fis, and HU) to DNA; 2) pairing of Hin- binding sites; 3) invertasome formation; 4) DNA strand cleavage; 5) strand rotation and religation. A series of topoisomers of the wild type DNA substrate plasmid (ranging from fully relaxed molecules to those with more than the physiological superhelical density (the physiological superhelical density of pKH336 from Escherichia coli DH10B is -0.072 in this study)) was generated, and the role of negative supercoiling in each step of the inversion reaction was investigated. We found differences in the dependence of the formation of paired Hin- binding sites and of the invertasome formation on the superhelical density of the substrate plasmid. Pairing of Hin-binding sites occurs independently from invertasome formation, and a relatively low degree of negative supercoiling is enough to promote maximal pairing. However, efficient invertasome formation requires higher levels of negative supercoiling.
|Additional Information:||Copyright © 1992 by the American Society for Biochemistry and Molecular Biology. (Received for publication, October 29, 1991) We thank Robert Bourret, David Steel, and Jacques J. Pene for help in preparing this manuscript. This work was supported by a grant from the National Science of Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.|
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|Deposited On:||15 Jan 2009 22:46|
|Last Modified:||26 Dec 2012 10:43|
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