title: A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement.
abstract: 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.
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Sona
authors.initials: S
authors.lastname: Valuchova
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Fulnecek
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Petrov
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Tripsianes
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic.
authors.forename: Karel
authors.initials: K
authors.lastname: Riha
mesh_terms: D016880:Anisotropy; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004261:DNA Replication; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007477:Ions; D000072200:Ku Autoantigen / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009696:Nucleic Acids / Q000737:chemistry*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
chemical_list: D005456:Fluorescent Dyes; D007477:Ions; D009696:Nucleic Acids; D011506:Proteins; D012313:RNA; D004247:DNA; D015245:Deoxyribonuclease BamHI; D000072200:Ku Autoantigen