title: Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.
abstract: Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect.
authors.affiliation: Department of Chemistry, University of Puerto Rico, Río Piedras Campus, Río Piedras, PR 00936, United States.
authors.forename: Mónica
authors.initials: M
authors.lastname: Fernández-Sierra
authors.affiliation: Department of Chemistry, University of Puerto Rico, Río Piedras Campus, Río Piedras, PR 00936, United States. Electronic address: edwin.quinones3@upr.edu.
authors.forename: Edwin
authors.initials: E
authors.lastname: Quiñones
mesh_terms: D001583:Benzoxazoles / Q000737:chemistry*; D001665:Binding Sites; D002412:Cations; D004247:DNA / Q000032:analysis / Q000737:chemistry; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors; D004277:DNA, Single-Stranded / Q000032:analysis / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000737:chemistry*; D007364:Intercalating Agents / Q000737:chemistry; D008956:Models, Chemical; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011806:Quinolinium Compounds / Q000737:chemistry*; D013696:Temperature
chemical_list: D001583:Benzoxazoles; D002412:Cations; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta; D003851:Deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases
keywords: DNA intercalator; Endonuclease; Exonuclease; Fluorescence; Kinetics; Single-stranded DNA binding protein