Publication details

A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement

Authors

VALUCHOVÁ Soňa FULNEČEK Jaroslav PETROV Alexander TRIPSIANES Konstantinos ŘÍHA Karel

Year of publication 2016
Type Article in Periodical
Magazine / Source Scientific Reports
MU Faculty or unit

Central European Institute of Technology

Citation
Web http://www.nature.com/articles/srep39653
Doi http://dx.doi.org/10.1038/srep39653
Field Biochemistry
Keywords SINGLE-MOLECULE; ENDONUCLEASE XPF-ERCC1; TRANSCRIPTION FACTORS; DNA INTERACTIONS; BINDING; REPAIR; POLYMERASE; INHIBITORS; ANISOTROPY; MECHANISM
Description Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions.
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