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
Volume43
Issue7
Paginatione43
Date Published2015 Apr 20
ISSN1362-4962
KeywordsDNA, DNA-Binding Proteins, Nucleic Acid Conformation, Protein Binding
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.

DOI10.1093/nar/gku1381
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