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Updated: May 20, 2026
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
Published on: February 17, 2011
Loss of BRCA1-A complex function in RAP80 null tumor cells
Chunjing Bian1, Rong Wu, Kathleen Cho
1Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Abstract:
Receptor Associated Protein 80 (RAP80) is a subunit of the BRCA1-A complex and targets BRCA1 to DNA damage sites in response to DNA double strand breaks. Since mutations of BRCA1 are associated with familial ovarian cancers, we screened 26 ovarian cancer-derived cell lines for RAP80 mutations and found that TOV-21G cells harbor a RAP80 mutation (c.1107G >A). This mutation generates a stop codon at Trp369, which deletes the partial AIR region and the C-terminal zinc fingers of RAP80. Interestingly, both the mutant and wild type alleles of RAP80 lose their expression due to promoter hypermethylation, suggesting that TOV-21G is a RAP80-null cell line. In these cells, not only is the BRCA1-A complex disrupted, but the relocation of the remaining subunits in the BRCA1-A complex including BRCA1, CCDC98, NBA1, BRCC36 and BRE is significantly suppressed. Moreover, TOV-21G cells are hypersensitive to ionizing radiation, which is due to the compromised DNA damage repair capacity in these cells. Reconstitution of TOV-21G cells with wild type RAP80 rescues these cellular defects in response to DNA damage. Thus, our results demonstrate that RAP80 is a scaffold protein in the BRCA1-A complex. Identification of TOV-21G as a RAP80 null tumor cell line will be very useful for the study of the molecular mechanism in DNA damage response.
Insights
Receptor Associated Protein 80 (RAP80) is crucial for DNA repair by scaffolding the BRCA1-A complex. A RAP80-null ovarian cancer cell line, TOV-21G, exhibits DNA repair defects and radiation hypersensitivity.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Receptor Associated Protein 80 (RAP80) is a key component of the BRCA1-A complex, essential for targeting BRCA1 to DNA double-strand break sites.
- BRCA1 mutations are linked to hereditary ovarian cancers, highlighting the importance of understanding DNA damage response pathways.
Purpose of the Study:
- To investigate the role of RAP80 in ovarian cancer by screening for mutations in RAP80 in ovarian cancer cell lines.
- To characterize the functional consequences of RAP80 loss in the identified RAP80-null TOV-21G cell line.
Main Methods:
- Screening of 26 ovarian cancer cell lines for RAP80 mutations.
- Mutation analysis, including identification of a specific mutation (c.1107G >A) in TOV-21G cells.
- Assessment of BRCA1-A complex integrity, subunit relocation, DNA damage repair capacity, and radiation sensitivity.
- Complementation assays by reintroducing wild-type RAP80 into TOV-21G cells.
Main Results:
- TOV-21G cells were identified to harbor a RAP80 mutation (c.1107G >A) leading to a premature stop codon and loss of functional domains.
- Both mutant and wild-type RAP80 alleles in TOV-21G cells showed hypermethylation-induced silencing, resulting in a RAP80-null state.
- The BRCA1-A complex was disrupted in TOV-21G cells, with suppressed relocation of its subunits to DNA damage sites.
- TOV-21G cells displayed hypersensitivity to ionizing radiation due to impaired DNA damage repair.
Conclusions:
- RAP80 functions as a critical scaffold protein within the BRCA1-A complex, facilitating DNA damage response.
- The RAP80-null TOV-21G cell line provides a valuable model for studying the molecular mechanisms of DNA damage response and its implications in ovarian cancer.
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