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

TitleA robust assay to measure DNA topology-dependent protein binding affinity.
Publication TypeJournal Article
Year of Publication2015
AuthorsLitwin, TR, Solà, M, Holt, IJ, Neuman, KC
JournalNucleic Acids Res
Date Published2015 Apr 20
KeywordsDNA, DNA-Binding Proteins, Nucleic Acid Conformation, Protein Binding

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.

Alternate JournalNucleic Acids Res.
Citation Key10.1093/nar/gku1381
PubMed ID25552413
PubMed Central IDPMC4402506
Grant ListMC_U105663140 / / Medical Research Council / United Kingdom
/ / Intramural NIH HHS / United States