Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions Journal Article

Authors: Saeki, H.; Siaud, N.; Christ, N.; Wiegant, W. W.; Van Buul, P. P. W.; Han, M.; Zdzienicka, M. Z.; Stark, J. M.; Jasin, M.
Article Title: Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions
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. © 2006 by The National Academy of Sciences of the USA.
Keywords: controlled study; nonhuman; protein domain; protein motif; animal cell; phenotype; mammalia; animals; mice; dna damage; homologous recombination; dna repair; gene expression; carboxy terminal sequence; protein binding; genetic variability; brca2 protein; chromosome aberration; tumor suppressor gene; genetic recombination; recombinant fusion proteins; recombination, genetic; chromosome aberrations; single stranded dna; dna, single-stranded; mutagenesis; replication protein a; cell mutant; rad51 protein; rad51 recombinase; cricetinae; double-strand break; brca2 gene; mammalian cells; rad51; brc repeat
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 103
Issue: 23
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 2006-06-06
Start Page: 8768
End Page: 8773
Language: English
DOI: 10.1073/pnas.0600298103
PUBMED: 16731627
PROVIDER: scopus
PMCID: PMC1482653
Notes: --- - "Cited By (since 1996): 40" - "Export Date: 4 June 2012" - "CODEN: PNASA" - "Source: Scopus"
Citation Impact
MSK Authors
  1. Hiroshi Saeki
    1 Saeki
  2. Jeremy M Stark
    8 Stark
  3. Nicole Christ
    9 Christ
  4. Nicolas Siaud
    4 Siaud
  5. Maria Jasin
    231 Jasin
  6. Mingguang Han
    5 Han