title: Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy.
abstract: We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ghoneim
authors.forename: Maria
authors.initials: M
authors.lastname: Spies
mesh_terms: D001105:Archaea / Q000737:chemistry / Q000378:metabolism*; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D019641:DNA, Archaeal / Q000378:metabolism*; D004867:Equipment Design; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004265:DNA Helicases
keywords: DNA damage recognition; DNA repair; XPD helicase; multicolor single-molecule detection; protein domain motion; total-internal reflection fluorescence microscopy