Format

Send to

Choose Destination
Nucleic Acids Res. 2013 Apr 1;41(6):3576-87. doi: 10.1093/nar/gkt056. Epub 2013 Feb 7.

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

Author information

1
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

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.

PMID:
23393192
PMCID:
PMC3616717
DOI:
10.1093/nar/gkt056
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center