1. Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase.
- Author
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Masuda-Ozawa T, Hoang T, Seo YS, Chen LF, and Spies M
- Subjects
- Binding Sites, DNA metabolism, DNA Helicases chemistry, DNA Replication, DNA-Binding Proteins chemistry, HEK293 Cells, Humans, Protein Structure, Tertiary, Rad51 Recombinase metabolism, DNA Helicases metabolism, DNA-Binding Proteins metabolism, Ubiquitination
- Abstract
DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box-containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.
- Published
- 2013
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