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Title

A robust assay to measure DNA topology-dependent protein binding affinity

AuthorsLitwin, Tamara R.; Solà, Maria ; Holt, Ian J.; Neuman, Keir C.
Issue Date18-Dec-2014
PublisherOxford University Press
CitationNucleic Acids Research 43(7): e43 (2014)
Abstract© 2015 Published by Oxford University Press on behalf of Nucleic Acids Research 2014. DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.
Publisher version (URL)http://dx.doi.org/10.1093/nar/gku1381
URIhttp://hdl.handle.net/10261/124202
DOI10.1093/nar/gku1381
Identifiersdoi: 10.1093/nar/gku1381
issn: 1362-4962
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