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

doi:10.1093/nar/gkt056

Back

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

Journal article

Open access

Peer reviewed

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 and Maria Spies

Nucleic acids research, Vol.41(6), pp.3576-3587

04/2013

DOI: 10.1093/nar/gkt056

PMCID: PMC3616717

PMID: 23393192

Files and links (1)

url

https://doi.org/10.1093/nar/gkt056View

Published (Version of record) Open Access

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.

Genome Integrity, Repair and

Details

Title: Subtitle: Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase
Creators: Tokiha Masuda-Ozawa - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Trish Hoang - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Yeon-Soo Seo - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Lin-Feng Chen - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Maria Spies - Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Resource Type: Journal article
Publication Details: Nucleic acids research, Vol.41(6), pp.3576-3587
DOI: 10.1093/nar/gkt056
PMID: 23393192
PMCID: PMC3616717
NLM abbreviation: Nucleic Acids Res
ISSN: 0305-1048
eISSN: 1362-4962
Publisher: Oxford University Press
Language: English
Date published: 04/2013
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984025265502771

Metrics

11 Record Views

30 Times Cited - Web of Science

See more details