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Multiple possible sites of BRCA2 interacting with DNA repair protein RAD51
T Katagiri1, H Saito, A Shinohara
1Department of Human Genome Analysis, Japanese Foundation for Cancer Research, Tokyo, Japan.
Abstract:
To investigate the biological consequences of aberrant BRCA2 protein during mammary carcinogenesis, we attempted to identify proteins that normally interact with BRCA2. By using a yeast two-hybrid system with a hybrid protein that contained residues 639-1,508 of BRCA2 protein fused to the GAL4 DNA-binding domain, we isolated five independent cDNA clones that encoded parts of RAD51 protein, a human homolog of bacterial RecA. In vitro experiments using anti-RAD51 antibody confirmed interaction of BRCA2 with RAD51. The RAD51-binding region of BRCA2 detected in the present study was distinct from the region reported recently. Further studies using smaller portions of BRCA2 defined at least two additional RAD51-binding domains, residues 982-1,066 and 1,139-1,266. Our results suggest that BRCA2 can interact with RAD51 through multiple sites of BRCA2 and that control of mitotic and meiotic recombination and/or of genomic integrity through binding to RAD51 may be a crucial mechanism by which BRCA2 suppresses abnormal proliferation of mammary cells.
Insights
This study identifies RAD51 protein as a binding partner for BRCA2, revealing multiple interaction sites. This interaction is crucial for BRCA2
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- BRCA2 protein plays a critical role in DNA repair and maintaining genomic stability.
- Aberrant BRCA2 function is implicated in mammary carcinogenesis.
- Identifying BRCA2 interacting proteins is key to understanding its biological consequences.
Purpose of the Study:
- To identify proteins that interact with BRCA2.
- To elucidate the role of BRCA2-protein interactions in mammary carcinogenesis.
- To map the RAD51-binding domains within the BRCA2 protein.
Main Methods:
- Yeast two-hybrid system to screen for BRCA2 interacting proteins.
- Utilizing a hybrid protein of BRCA2 (residues 639-1,508) fused to GAL4 DNA-binding domain.
- In vitro experiments with anti-RAD51 antibody and smaller BRCA2 fragments.
Main Results:
- RAD51 protein, a human homolog of bacterial RecA, was identified as a BRCA2 interacting protein.
- In vitro assays confirmed the interaction between BRCA2 and RAD51.
- Multiple RAD51-binding domains were identified in BRCA2, including residues 982-1,066 and 1,139-1,266.
Conclusions:
- BRCA2 interacts with RAD51 through multiple binding sites.
- BRCA2-RAD51 interaction is essential for controlling recombination and genomic integrity.
- This mechanism likely contributes to BRCA2's role in suppressing abnormal mammary cell proliferation.