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Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
Published on: February 17, 2011
BRCA1-associated protein 1 interferes with BRCA1/BARD1 RING heterodimer activity
Hiroyuki Nishikawa1, Wenwen Wu, Ayaka Koike
1Division of Breast and Endocrine Surgery, Institute of Advanced Medical Science, and Department of Radiology, St. Marianna University School of Medicine, Kawasaki, Japan.
Cancer Research
|January 2, 2009
Summary
BRCA1-associated protein 1 (BAP1) inhibits BRCA1/BARD1 E3 ligase activity, impacting DNA damage response and cell cycle progression. BAP1 plays a crucial role in regulating ubiquitination pathways critical for genomic stability.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- BRCA1, a tumor suppressor, forms a RING heterodimer E3 ligase with BARD1.
- BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that interacts with BRCA1.
- The precise role of BAP1 in the E3 ligase activity of BRCA1/BARD1 remains unclear.
Purpose of the Study:
- To elucidate the mechanism by which BAP1 influences the E3 ligase activity of the BRCA1/BARD1 complex.
- To investigate the functional consequences of BAP1's interaction with BRCA1/BARD1 in cellular processes.
- To determine BAP1's role in DNA damage response and cell cycle regulation.
Main Methods:
- Protein interaction studies using domains of BAP1 (residues 182-365) and the BARD1 RING finger domain.
- Surface plasmon resonance (BIAcore) to analyze BAP1's effect on BRCA1/BARD1 association.
- In vitro ubiquitination assays with BRCA1/BARD1 and BAP1 (wild-type and C91S mutant) on substrates like NPM1/B23.
- In vivo studies using short hairpin RNA (shRNA) to inhibit BAP1 expression and assess cellular responses to ionizing irradiation and cell cycle progression.
Main Results:
- BAP1 directly interacts with BARD1, inhibiting the E3 ligase activity of the BRCA1/BARD1 complex.
- BAP1 binding disrupts the BRCA1/BARD1 association, leading to reduced BRCA1 autoubiquitination and NPM1/B23 ubiquitination.
- BAP1 exhibits in vitro deubiquitinating activity and also inhibits ubiquitination via a catalytically independent mechanism.
- Depletion of BAP1 sensitizes cells to ionizing irradiation and causes S-phase progression defects.
Conclusions:
- BAP1 acts as a negative regulator of BRCA1/BARD1 E3 ligase activity through direct interaction with BARD1.
- BAP1 employs dual mechanisms: enzymatic deubiquitination and non-enzymatic inhibition of ubiquitination.
- BAP1 and BRCA1/BARD1 function coordinately in regulating ubiquitination for DNA damage response and cell cycle control.
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