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Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
BRCA1 ubiquitinates RPB8 in response to DNA damage
Wenwen Wu1, Hiroyuki Nishikawa, Ryosuke Hayami
1Division of Breast and Endocrine Surgery, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan.
Cancer Research
|February 7, 2007
Summary
The BRCA1-BARD1 complex polyubiquitinates RNA polymerase subunit RPB8, enhancing cell survival after DNA damage. This ubiquitination increases soluble RPB8 and is crucial for DNA repair pathways.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The BRCA1-BARD1 complex is known to catalyze non-proteolytic polyubiquitin chains, but its precise mechanisms remain unclear.
- Understanding BRCA1's function is critical due to its role as a tumor suppressor in breast and ovarian cancers.
Purpose of the Study:
- To investigate the role of BRCA1-BARD1 in response to DNA damage.
- To identify novel substrates and mechanisms regulated by BRCA1-BARD1 activity.
Main Methods:
- Proteomics screening to identify proteins modified by BRCA1 after DNA damage.
- In vivo and in vitro ubiquitination assays using BRCA1-BARD1 and RPB8.
- RNA interference to assess BRCA1's role in RPB8 ubiquitination.
- Site-directed mutagenesis to create ubiquitin-resistant RPB8.
- Cell viability assays and caspase activity measurements.
Main Results:
- RPB8, a subunit of RNA polymerases, was identified as a BRCA1-BARD1 substrate following epirubicin treatment.
- BRCA1-BARD1 mediated RPB8 polyubiquitination in vitro and in vivo, increasing soluble RPB8 without causing degradation.
- RPB8 ubiquitination occurred rapidly after UV irradiation and was dependent on BRCA1.
- Ubiquitin-resistant RPB8 mutants led to UV hypersensitivity and increased caspase activity in HeLa cells.
Conclusions:
- BRCA1-BARD1-mediated ubiquitination of RPB8 is a novel mechanism involved in DNA damage response.
- This ubiquitination pathway promotes cell survival by maintaining RNA polymerase function and integrity.
- Targeting this pathway could offer new therapeutic strategies for BRCA1-deficient cancers.
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