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

Hiroshi Saeki, Nicolas Siaud, Nicole Christ, Wouter W. Wiegant, Paul P.W. Van Buul, Mingguang Han, Małgorzata Z. Zdzienicka, Jeremy M. Stark, Maria Jasin

Research output: Contribution to journalArticle

70 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)8768-8773
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number23
DOIs
Publication statusPublished - Jun 6 2006

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Replication Protein A
DNA Repair
Proteins
DNA Damage
Rad51 Recombinase
Homologous Recombination
Protein Subunits
Chromosome Aberrations
Carrier Proteins
Mutation
Survival
DNA
Neoplasms

All Science Journal Classification (ASJC) codes

  • General

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Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions. / Saeki, Hiroshi; Siaud, Nicolas; Christ, Nicole; Wiegant, Wouter W.; Van Buul, Paul P.W.; Han, Mingguang; Zdzienicka, Małgorzata Z.; Stark, Jeremy M.; Jasin, Maria.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 23, 06.06.2006, p. 8768-8773.

Research output: Contribution to journalArticle

Saeki, H, Siaud, N, Christ, N, Wiegant, WW, Van Buul, PPW, Han, M, Zdzienicka, MZ, Stark, JM & Jasin, M 2006, 'Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions', Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 23, pp. 8768-8773. https://doi.org/10.1073/pnas.0600298103
Saeki, Hiroshi ; Siaud, Nicolas ; Christ, Nicole ; Wiegant, Wouter W. ; Van Buul, Paul P.W. ; Han, Mingguang ; Zdzienicka, Małgorzata Z. ; Stark, Jeremy M. ; Jasin, Maria. / Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions. In: Proceedings of the National Academy of Sciences of the United States of America. 2006 ; Vol. 103, No. 23. pp. 8768-8773.
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