title: A robust assay to measure DNA topology-dependent protein binding affinity.
abstract: 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.
authors.affiliation: National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20814, USA Mitochondrial Biology Unit, Medical Research Council, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.
authors.forename: Tamara R
authors.initials: TR
authors.lastname: Litwin
authors.affiliation: Department of Structural Biology, Molecular Biology Institute of Barcelona (CSIC), 08028 Barcelona, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Solà
authors.affiliation: National Institute for Medical Research, Medical Research Council, The Ridgeway, Mill Hill, London, NW7 1AA, UK.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Holt
authors.affiliation: National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20814, USA neumankc@mail.nih.gov.
authors.forename: Keir C
authors.initials: KC
authors.lastname: Neuman
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA