Saeki, H. et al. Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions. Proc. Natl Acad. Sci. USA 103, 8768-8773

Nicolaus Copernicus University, Toruń, Kujawsko-Pomorskie, Poland
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2006; 103(23):8768-73. DOI: 10.1073/pnas.0600298103
Source: PubMed


The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.

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Available from: Wouter Wiegant, Nov 05, 2014
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    • "associated BRCA2 mutations that compromise inter - actions with RAD51 have been found within BRC repeats ( Venkitaraman , 2009 ) . Much of our knowledge of BRCA2 function comes from studying fragments of the BRCA2 protein or investigating BRCA2 orthologues , such as Brh2 in Ustilago maydis and BRC2 in Caenorhabditis elegans ( Yang et al . , 2005 ; Saeki et al . , 2006 ; Carreira et al . , 2009 ) . Details of mammalian BRCA2 functions are still not known due to difficulties in isolating the full - length protein , which is composed of 3418 amino acids in humans . Recently , however , three independent groups have successfully purified and func - tionally validated full - length human BRCA2 from human "
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    • "The BRC–RPA fusion protein functioned as efficiently as full-length BRCA2 in response to MMS, and it provided partial complementation of BLE sensitivity, indicating that repair can be catalysed in the absence of BRCA2 C-terminal sequence. This is consistent with the ability of this fusion to support RAD51 foci formation after BLE treatment in T. brucei (27) and, more broadly, with the functioning of a BRC–RPA fusion in response to ultraviolet irradiation in U. maydis (47) and in response to mitomycin C or ionising radiation damage in mammals (23). Neither of the BRCA2 variants with only one BRC repeat supported fully efficient repair. "
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    • "We therefore sought to address whether FANCD2 acts in synergy with RAD51 when protecting stalled forks or whether they act epistatically , within a common pathway. To test this, we expressed the BRC4 peptide (Saeki et al., 2006), which suppresses DNA binding of RAD51 and thus disrupts RAD51 filaments. "
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