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: 9.67). 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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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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    ABSTRACT: When the glucose supply is high, despite the presence of oxygen Saccharomyces cerevisiae uses fermentation as its main metabolic pathway and switches to oxidative metabolism only when this carbon source is limited. There are similarities between glucose-induced repression of oxidative metabolism of yeast and metabolic reprogramming of tumor cells. The glucose-induced repression of oxidative metabolism is regulated by oncogene homologues in yeast, such as Ras and Sch9p, the yeast homologue of Akt. Yeast also undergoes an apoptosis-like programmed cell death process sharing several features with mammalian apoptosis, including oxidative stress and a major role played by mitochondria. Evasion of apoptosis and sustained proliferative signalling are hallmarks of cancer. This, together with the possibility of heterologous expression of human genes in yeast, has allowed new insights to be obtained into the function of mammalian oncogenes/oncosuppressors. Here we elaborate on the similarities between tumor and yeast cells underpinning the use of this model organism in cancer research. We also review the achievements obtained through heterologous expression in yeast of p53, BRCA1 and BRCA2 which are among the best known cancer susceptibility genes, with the aim of understanding their role in tumorigenesis. Yeast cell-based functional assays for cancer genetic testing will also be dealt with. This article is protected by copyright. All rights reserved.
    FEMS Yeast Research 09/2013; 14(1). DOI:10.1111/1567-1364.12094 · 2.82 Impact Factor
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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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    ABSTRACT: Trypanosoma brucei survives in mammals through antigenic variation, which is driven by RAD51-directed homologous recombination of Variant Surface Glycoproteins (VSG) genes, most of which reside in a subtelomeric repository of >1000 silent genes. A key regulator of RAD51 is BRCA2, which in T. brucei contains a dramatic expansion of a motif that mediates interaction with RAD51, termed the BRC repeats. BRCA2 mutants were made in both tsetse fly-derived and mammal-derived T. brucei, and we show that BRCA2 loss has less impact on the health of the former. In addition, we find that genome instability, a hallmark of BRCA2 loss in other organisms, is only seen in mammal-derived T. brucei. By generating cells expressing BRCA2 variants with altered BRC repeat numbers, we show that the BRC repeat expansion is crucial for RAD51 subnuclear dynamics after DNA damage. Finally, we document surprisingly limited co-localization of BRCA2 and RAD51 in the T. brucei nucleus, and we show that BRCA2 mutants display aberrant cell division, revealing a function distinct from BRC-mediated RAD51 interaction. We propose that BRCA2 acts to maintain the huge VSG repository of T. brucei, and this function has necessitated the evolution of extensive RAD51 interaction via the BRC repeats, allowing re-localization of the recombinase to general genome damage when needed.
    Nucleic Acids Research 12/2012; 41(2). DOI:10.1093/nar/gks1192 · 9.11 Impact Factor
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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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    ABSTRACT: Genes mutated in patients with Fanconi anemia (FA) interact with the DNA repair genes BRCA1 and BRCA2/FANCD1 to suppress tumorigenesis, but the molecular functions ascribed to them cannot fully explain all of their cellular roles. Here, we show a repair-independent requirement for FA genes, including FANCD2, and BRCA1 in protecting stalled replication forks from degradation. Fork protection is surprisingly rescued in FANCD2-deficient cells by elevated RAD51 levels or stabilized RAD51 filaments. Moreover, FANCD2-mediated fork protection is epistatic with RAD51 functions, revealing an unanticipated fork protection pathway that connects FA genes to RAD51 and the BRCA1/2 breast cancer suppressors. Collective results imply a unified molecular mechanism for repair-independent functions of FA, RAD51, and BRCA1/2 proteins in preventing genomic instability and suppressing tumorigenesis.
    Cancer cell 07/2012; 22(1):106-16. DOI:10.1016/j.ccr.2012.05.015 · 23.52 Impact Factor
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