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

Tokiha Masuda-Ozawa, Trish Hoang, Yeon Soo Seo, Lin Feng Chen, Maria Spies

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)3576-3587
Number of pages12
JournalNucleic acids research
Volume41
Issue number6
DOIs
StatePublished - Apr 2013

ASJC Scopus subject areas

  • Genetics

Fingerprint

Dive into the research topics of 'Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase'. Together they form a unique fingerprint.

Cite this