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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
Published on: January 29, 2018
BLM SUMOylation regulates ssDNA accumulation at stalled replication forks
Karen J Ouyang1, Mary K Yagle, Michael J Matunis
1Department of Medicine, University of Chicago Chicago, IL, USA.
Frontiers in Genetics
|September 13, 2013
Summary
SUMOylation of BLM helicase regulates RAD51 accumulation at stalled replication forks by controlling single-stranded DNA (ssDNA) binding protein RPA. This ensures proper homologous recombination (HR) repair of DNA double-strand breaks (DSBs).
Area of Science:
- Molecular Biology
- DNA Replication and Repair
- Cellular Stress Response
Background:
- Polymerase stalling generates single-stranded DNA (ssDNA), leading to replication fork instability and double-strand breaks (DSBs).
- RAD51 protein is crucial for stabilizing stalled forks and initiating homologous recombination (HR) repair.
- SUMO modification of the BLM helicase is a recently identified requirement for RAD51 accumulation at stalled forks.
Purpose of the Study:
- To investigate how BLM helicase SUMOylation controls RAD51 accumulation at stalled replication forks.
- To characterize the role of HR proteins and RPA in cells with normal (BLM+) versus SUMO-mutant (SM-BLM) BLM.
Main Methods:
- Analysis of HR proteins (BRCA2, RAD52) and RPA accumulation in HU-treated BLM+ and SM-BLM cells.
- In vitro helicase activity assays of SUMOylated and unSUMOylated BLM.
- In vitro binding assays and immunoprecipitation to assess BLM-RPA complex formation.
Main Results:
- SM-BLM cells showed impaired BRCA2 and RAD52 accumulation and excess RPA at stalled forks.
- BLM SUMOylation did not affect BLM helicase activity or direct BLM-RPA binding but regulated BLM-RPA complex formation.
- BLM SUMOylation controls the extent of ssDNA accumulation during polymerase stalling.
Conclusions:
- BLM SUMOylation acts as a licensing mechanism to permit and regulate HR at damaged replication forks.
- This regulation involves controlling the dynamic interplay between RPA and HR factors at stalled forks.
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