Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase

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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.
Publisher
OXFORD UNIV PRESS
Issue Date
2013-04
Language
English
Article Type
Article
Keywords

BOX DNA HELICASE; HOMOLOGOUS RECOMBINATION; STRAND EXCHANGE; HUMAN RAD51; REPLICATION FORKS; ATPASE ACTIVITY; PROTEIN; BINDING; FILAMENTS; REPAIR

Citation

NUCLEIC ACIDS RESEARCH, v.41, no.6, pp.3576 - 3587

ISSN
0305-1048
DOI
10.1093/nar/gkt056
URI
http://hdl.handle.net/10203/174196
Appears in Collection
BS-Journal Papers(저널논문)
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