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. Interac

SUBMITTER: Ghoneim M 

PROVIDER: S-EPMC4189620 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature