title: Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase.
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.
authors: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors: Tokiha
authors: T
authors: Masuda-Ozawa
authors: Trish
authors: T
authors: Hoang
authors: Yeon-Soo
authors: YS
authors: Seo
authors: Lin-Feng
authors: LF
authors: Chen
authors: Maria
authors: M
authors: Spies
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; D051135:Rad51 Recombinase; D004265:DNA Helicases; C464443:FBH1 protein, human
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