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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

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 07/2006; 103(23):8768-73. DOI: 10.1073/pnas.0600298103
Source: PubMed

ABSTRACT 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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    • "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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    • "Previous structural analyses of cancer-associated mutations affecting the BRC repeats revealed that weakening RAD51 affinity in one repeat is enough to increase breast cancer susceptibility [10] [29] [30]. However, the restoration of BRCA2 function in BRCA2- deficient cells by the expression of the construct connecting a single BRC repeat and the replication protein A large subunit, which delivers RAD51 to single-stranded DNA [31], suggests that further analysis is necessary to confirm a causal relationship between this mutation and breast cancer onset. Although the effects of random or cancer-associated amino acid substitutions in human BRC4 on the inhibition of RAD51 interactions are reported [23] [30], there are no reports about the effect of amino acid substitutions among animal species. "
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