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Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy
Journal article   Open access   Peer reviewed

Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy

Mohamed Ghoneim and Maria Spies
Nano letters, Vol.14(10), pp.5920-5931
10/08/2014
DOI: 10.1021/nl502890g
PMCID: PMC4189620
PMID: 25204359
url
https://doi.org/10.1021/nl502890gView
Published (Version of record) Open Access

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.
 Archaeal Proteins - metabolism Protein Structure, Tertiary DNA Helicases - chemistry Archaeal Proteins - chemistry Archaea - chemistry DNA, Archaeal - metabolism Equipment Design Microscopy, Fluorescence - instrumentation DNA Helicases - metabolism Archaea - metabolism DNA Repair Protein Binding Binding Sites

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