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BRCA2 Keeps Rad51 in Line
Molecular Cell 1262
BRCA2 Keeps Rad51 in Line: High-Fidelity Homologous Recombination Prevents Breast and Ovarian Cancer? The mechanistic link between the BRCA2 protein, homologous recombination, and genomic instability has become clearer as a result of recent work describing the structures of different BRCA2 domains bound to Rad51 and to single-stranded DNA. Familial breast and ovarian cancer can be triggered by a mutation in one allele of either BRCA1 or BRCA2. The sequence of events that occur from the initial germline mutation to the development of cancer in breast or ovarian epithelium is unclear, but loss of function of the second allele appears to be required. However, if both alleles are knocked out in a developing mouse, or even in cells, there is failure to develop or to proliferate. Solving this “tumor suppressor paradox” is probably the key to understanding how loss of BRCA1 or BRCA2 function predisposes to breast and ovarian cancer. Defining the relevant physiological function of BRCA1 has proven to be extremely complicated; studies have ascribed a role for BRCA1 in an overwhelming number of processes, including DNA replication, cell cycle checkpoint control, DNA repair, regulation of transcription, protein ubiquitination, apoptosis, and chromatin remodeling. How all these activities might be unified is unclear. On the other hand, there has been only one function delineated so far for BRCA2—as a cofactor in homologous recombination that requires its interaction with Rad51 (Davies et al., 2001; Xia et al., 2001). As such, the role of BRCA2 in regulating Rad51-dependent homologous recombination may be the key to understanding the cancer-prone phenotype of both BRCA1 and BRCA2 mutations. Two recent publications have highlighted structural aspects of BRCA2, and both studies offer some insight into the structural basis of BRCA2’s role in recombinational repair. Rad51 interacts with the BRC repeat regions of BRCA2, encoded by the very large exon 11. There are eight BRC repeats, and BRC3 and BRC4 have the strongest interaction with Rad51. Pellegrini et al. (2002) have solved the crystal structure of the BRC4 peptide sequence (amino acids 1517 to 1551) of BRCA2 bound to Rad51 (⌬1-96). This structure demonstrates a series of hydrophobic and hydrophilic interactions involving the hairpin structure of BRC4 created by the short anti-parallel  sheets and an ␣ helix (see Figure). Contact between BRC4 and Rad51 is maintained from Leu1521 to Glu1548 of the BRC4 sequence. The structure of the BRC4 peptide is similar to the monomermonomer interaction domain of RecA (the bacterial homolog of Rad51) that is involved in filament formation. Other evidence suggests that the BRC4 peptide can inhibit Rad51 filament formation, by blocking Rad51 monomer interactions. Whereas GFP-tagged wild-type Rad51 protein can interact with endogenous Rad51, a GFP-tagged Rad51 with mutations (F86E and A89E) in
the monomer interaction site retains the ability to bind BRCA2, but not endogenous Rad51. In addition, expression of BRC4 (or BRC3) peptides in cells expressing GFP-tagged Rad51 blocks the ability of Rad51 to form nuclear foci (presupposing that filament formation and focus formation go hand-in-hand). Previous in vitro work had suggested that BRC3 or BRC4 peptides could inhibit the formation of Rad51 complexes (Davies et al., 2001). However, in living cells, the effect of full-length BRCA2 is to promote homologous recombination (Moynahan et al., 2001); expression of the BRC4 peptide inhibits BRCA2-stimulated homologous recombination (Xia et al., 2001); and, the formation of Rad51 foci appears to require BRCA2 (and BRCA1), especially after exposure to ionizing radiation (S.C. West, personal communication; S.N.P., unpublished data). These observations present a quandary: why do BRC3 and BRC4 peptides inhibit filament formation, but the complete BRCA2 protein appears to promote filament formation? One possibility is that the use of the BRC4 peptide presents an excess of the Rad51 interacting moiety, whereas the location of BRC4 (and other BRC repeats) within the whole BRCA2 protein may dictate a more highly controlled interaction with Rad51. Thus, the quandary may be resolved by proposing a model of BRCA2 as a loading factor for Rad51 on ssDNA by means of its BRC peptide interaction. As the Rad51 bound to BRCA2 finds itself positioned adjacent to a Rad51 monomer on ssDNA, the monomer-monomer interaction becomes favored over the BRCA2 interaction (see Figure). This model is unsubstantiated but summarizes our current knowledge of how these proteins might act. We know that Rad51-dependent homologous recombination and Rad51 filament formation in S phase do occur in the absence of BRCA2. However, the presence of BRCA2 is required to prevent major errors, such as chromosome aberrations. Whether these errors reflect utilization of an alternative pathway of repair, such as nonhomologous end joining (Moynahan et al., 2001), or occur as a result of error-prone homology directed repair (Tutt et al., 2001) remains to be solved. The dilemmas posed by the Pellegrini work (Pellegrini et al., 2002) only serve to remind us that the BRCA2 protein is extremely large, with many domains that have been shown to be important in cancer prevention but whose function is unknown. The second paper providing structural insights into the function of the BRCA2 protein (Yang et al., 2002) solved the crystal structure of the C-terminal 800 amino acids of BRCA2, completely separate from the BRC repeat region of the protein, bound to ssDNA (an oligo dT sequence). The presence of DSS1 (deleted in split-hand, split-foot syndrome), a protein found to interact with BRCA2 in a yeast two-hybrid screen, was required to make the complex soluble and to promote crystal formation. The BRCA2 structure delineated in this study reveals a series of oligonucleotide binding folds (OB1/OB2/OB3) that bind ssDNA, and a “tower” region between OB2 and OB3 containing a three-helix bundle with a structure very similar to the Hin recombinase. The structure of the OB2/OB3 domains has significant alignment with replication protein A (RPA), an ssDNA binding protein. Two tempting models are raised by these observations. In one, BRCA2 could play a role in displacing RPA from ssDNA, and
Previews 1263
The Role of BRCA2 in Rad51-Dependent Homologous Recombination Double-strand breaks that are repaired by homologous recombination undergo Resection of the 5⬘ ends, with RPA binding to the 3⬘ ends. Conversion of the RPA-coated ssDNA to a Rad51 filament is necessary to initiate the strand exchange reaction. BRCA2 and Rad51 (and many other proteins, such as Rad51B/C/D and XRCC2/3) localize to the sites of DSBs, probably with a predilection for a ds/ssDNA transition, which can be visualized as nuclear protein foci. Rad51 interacts with the BRC repeat region of BRCA2, which is shown as the violet colored projections that are maintained by the yellow antiparallel  sheets and the green ␣ helix. The proposed model is that the BRC repeat provides an assembly line of Rad51 monomers and that the C terminus of BRCA2 binds ssDNA, providing a means to displace RPA and allow the orderly assembly of the Rad51 filament. In the absence of BRCA2, critical events in the initiation of homologous recombination are impaired, and repair (and replication) errors accrue rapidly with each cell cycle.
that the essential role of the BRCA1 and BRCA2 “pathway” is in chromatin remodeling, with the effects on DNA repair being indirect, are weakened by these new studies. Both papers have shown that known BRCA2 mutations in cancer families map to critical regions of the interaction with Rad51 (exon 11, BRC repeats) and with ssDNA (C terminus). When mutations lead to a truncation in a protein, it is reasonably assumed that this affects its function. BRCA2 mutations found in patients with breast or ovarian cancer often lead to difficulties in interpretation, however, because it is not certain that a missense mutation produces a functional deficit. Functional assays of BRCA2 missense mutations are not readily available, but these new structural data may allow modeling and interpretation of how a mutation affects critical interaction sites. Returning to the question of how cancer arises in cells with deficiencies in BRCA2 (or BRCA1), it seems likely that the impaired function of homologous recombination is a key step. A “mutator” phenotype can be triggered by a defect in the regulation of homologous recombination, either by making the process error-prone or by shunting repair events into a nonhomologous end-joining pathway that is inherently mutagenic. Additional mutational steps, to bypass the proliferative block, are also necessary to allow a net growth in the number of cancer cells. However, what remains is the unsolved mystery of why this described defect in homologous recombination leads to the tissue-specific cancers of the breast and ovary. Proliferation in breast or ovarian epithelium may be associated with higher levels of endogenous DNA damage, relative to other tissues, which leads to replication stalling and requires homologous recombination for replication restart. Research into BRCA1 and BRCA2 function is now at a fever pitch: the next few years should be productive times in dissecting the function of these interesting large proteins. Simon N. Powell, Henning Willers, and Fen Xia Department of Radiation Oncology Massachusetts General Hospital and Harvard Medical School Building 149, 13th Street Charlestown, Massachusetts 02129 Selected Reading
allowing the formation of a Rad51 filament; alternatively, BRCA2 could primarily bind ssDNA and directly facilitate the formation of Rad51 filaments. The role of the “tower” region of the C terminus of BRCA2, with the recombinase motif, is more difficult to rationalize with the displacement and loading model, unless the loading of Rad51, filament formation, and strand exchange are interlinked processes, with BRCA2 being the linking protein (see Figure). This model was partly suggested in a recent editorial (Wilson and Elledge, 2002), in which BRCA2 was proposed to facilitate rate of loading of Rad51 and the organization of the Rad51 filament. The recent structural data strongly support the view that the primary role of BRCA2 is to facilitate the mechanism of homologous recombination. Prior suggestions
Davies, A.A., Masson, J.Y., McIlwraith, M.J., Stasiak, A.Z., Stasiak, A., Venkitaraman, A.R., and West, S.C. (2001). Mol. Cell 7, 273–282. Moynahan, M.E., Pierce, A.J., and Jasin, M. (2001). Mol. Cell 7, 263–272. Pellegrini, L., Yu, D.S., Lo, T., Anand, S., Lee, M., Blundell, T.L., and Venkitaraman, A.R. (2002). Nature 420, 287–293. Tutt, A., Bertwistle, D., Valentine, J., Gabriel, A., Swift, S., Ross, G., Griffin, C., Thacker, J., and Ashworth, A. (2001). EMBO J. 20, 4704–4716. Wilson, J.H., and Elledge, S.J. (2002). Science 297, 1822–1823. Xia, F., Taghian, D.G., DeFrank, J.S., Zeng, Z.C., Willers, H., Iliakis, G., and Powell, S.N. (2001). Proc. Natl. Acad. Sci. USA 98, 8644– 8649. Yang, H., Jeffrey, P.D., Miller, J., Kinnucan, E., Sun, Y., Thoma, N.H., Zheng, N., Chen, P.L., Lee, W.H., and Pavletich, N.P. (2002). Science 297, 1837–1848.
BRCA2 Keeps Rad51 in Line: High-Fidelity Homologous Recombination Prevents Breast and Ovarian Cancer? The mechanistic link between the BRCA2 protein, homologous recombination, and genomic instability has become clearer as a result of recent work describing the structures of different BRCA2 domains bound to Rad51 and to single-stranded DNA. Familial breast and ovarian cancer can be triggered by a mutation in one allele of either BRCA1 or BRCA2. The sequence of events that occur from the initial germline mutation to the development of cancer in breast or ovarian epithelium is unclear, but loss of function of the second allele appears to be required. However, if both alleles are knocked out in a developing mouse, or even in cells, there is failure to develop or to proliferate. Solving this “tumor suppressor paradox” is probably the key to understanding how loss of BRCA1 or BRCA2 function predisposes to breast and ovarian cancer. Defining the relevant physiological function of BRCA1 has proven to be extremely complicated; studies have ascribed a role for BRCA1 in an overwhelming number of processes, including DNA replication, cell cycle checkpoint control, DNA repair, regulation of transcription, protein ubiquitination, apoptosis, and chromatin remodeling. How all these activities might be unified is unclear. On the other hand, there has been only one function delineated so far for BRCA2—as a cofactor in homologous recombination that requires its interaction with Rad51 (Davies et al., 2001; Xia et al., 2001). As such, the role of BRCA2 in regulating Rad51-dependent homologous recombination may be the key to understanding the cancer-prone phenotype of both BRCA1 and BRCA2 mutations. Two recent publications have highlighted structural aspects of BRCA2, and both studies offer some insight into the structural basis of BRCA2’s role in recombinational repair. Rad51 interacts with the BRC repeat regions of BRCA2, encoded by the very large exon 11. There are eight BRC repeats, and BRC3 and BRC4 have the strongest interaction with Rad51. Pellegrini et al. (2002) have solved the crystal structure of the BRC4 peptide sequence (amino acids 1517 to 1551) of BRCA2 bound to Rad51 (⌬1-96). This structure demonstrates a series of hydrophobic and hydrophilic interactions involving the hairpin structure of BRC4 created by the short anti-parallel  sheets and an ␣ helix (see Figure). Contact between BRC4 and Rad51 is maintained from Leu1521 to Glu1548 of the BRC4 sequence. The structure of the BRC4 peptide is similar to the monomermonomer interaction domain of RecA (the bacterial homolog of Rad51) that is involved in filament formation. Other evidence suggests that the BRC4 peptide can inhibit Rad51 filament formation, by blocking Rad51 monomer interactions. Whereas GFP-tagged wild-type Rad51 protein can interact with endogenous Rad51, a GFP-tagged Rad51 with mutations (F86E and A89E) in
the monomer interaction site retains the ability to bind BRCA2, but not endogenous Rad51. In addition, expression of BRC4 (or BRC3) peptides in cells expressing GFP-tagged Rad51 blocks the ability of Rad51 to form nuclear foci (presupposing that filament formation and focus formation go hand-in-hand). Previous in vitro work had suggested that BRC3 or BRC4 peptides could inhibit the formation of Rad51 complexes (Davies et al., 2001). However, in living cells, the effect of full-length BRCA2 is to promote homologous recombination (Moynahan et al., 2001); expression of the BRC4 peptide inhibits BRCA2-stimulated homologous recombination (Xia et al., 2001); and, the formation of Rad51 foci appears to require BRCA2 (and BRCA1), especially after exposure to ionizing radiation (S.C. West, personal communication; S.N.P., unpublished data). These observations present a quandary: why do BRC3 and BRC4 peptides inhibit filament formation, but the complete BRCA2 protein appears to promote filament formation? One possibility is that the use of the BRC4 peptide presents an excess of the Rad51 interacting moiety, whereas the location of BRC4 (and other BRC repeats) within the whole BRCA2 protein may dictate a more highly controlled interaction with Rad51. Thus, the quandary may be resolved by proposing a model of BRCA2 as a loading factor for Rad51 on ssDNA by means of its BRC peptide interaction. As the Rad51 bound to BRCA2 finds itself positioned adjacent to a Rad51 monomer on ssDNA, the monomer-monomer interaction becomes favored over the BRCA2 interaction (see Figure). This model is unsubstantiated but summarizes our current knowledge of how these proteins might act. We know that Rad51-dependent homologous recombination and Rad51 filament formation in S phase do occur in the absence of BRCA2. However, the presence of BRCA2 is required to prevent major errors, such as chromosome aberrations. Whether these errors reflect utilization of an alternative pathway of repair, such as nonhomologous end joining (Moynahan et al., 2001), or occur as a result of error-prone homology directed repair (Tutt et al., 2001) remains to be solved. The dilemmas posed by the Pellegrini work (Pellegrini et al., 2002) only serve to remind us that the BRCA2 protein is extremely large, with many domains that have been shown to be important in cancer prevention but whose function is unknown. The second paper providing structural insights into the function of the BRCA2 protein (Yang et al., 2002) solved the crystal structure of the C-terminal 800 amino acids of BRCA2, completely separate from the BRC repeat region of the protein, bound to ssDNA (an oligo dT sequence). The presence of DSS1 (deleted in split-hand, split-foot syndrome), a protein found to interact with BRCA2 in a yeast two-hybrid screen, was required to make the complex soluble and to promote crystal formation. The BRCA2 structure delineated in this study reveals a series of oligonucleotide binding folds (OB1/OB2/OB3) that bind ssDNA, and a “tower” region between OB2 and OB3 containing a three-helix bundle with a structure very similar to the Hin recombinase. The structure of the OB2/OB3 domains has significant alignment with replication protein A (RPA), an ssDNA binding protein. Two tempting models are raised by these observations. In one, BRCA2 could play a role in displacing RPA from ssDNA, and
Previews 1263
The Role of BRCA2 in Rad51-Dependent Homologous Recombination Double-strand breaks that are repaired by homologous recombination undergo Resection of the 5⬘ ends, with RPA binding to the 3⬘ ends. Conversion of the RPA-coated ssDNA to a Rad51 filament is necessary to initiate the strand exchange reaction. BRCA2 and Rad51 (and many other proteins, such as Rad51B/C/D and XRCC2/3) localize to the sites of DSBs, probably with a predilection for a ds/ssDNA transition, which can be visualized as nuclear protein foci. Rad51 interacts with the BRC repeat region of BRCA2, which is shown as the violet colored projections that are maintained by the yellow antiparallel  sheets and the green ␣ helix. The proposed model is that the BRC repeat provides an assembly line of Rad51 monomers and that the C terminus of BRCA2 binds ssDNA, providing a means to displace RPA and allow the orderly assembly of the Rad51 filament. In the absence of BRCA2, critical events in the initiation of homologous recombination are impaired, and repair (and replication) errors accrue rapidly with each cell cycle.
that the essential role of the BRCA1 and BRCA2 “pathway” is in chromatin remodeling, with the effects on DNA repair being indirect, are weakened by these new studies. Both papers have shown that known BRCA2 mutations in cancer families map to critical regions of the interaction with Rad51 (exon 11, BRC repeats) and with ssDNA (C terminus). When mutations lead to a truncation in a protein, it is reasonably assumed that this affects its function. BRCA2 mutations found in patients with breast or ovarian cancer often lead to difficulties in interpretation, however, because it is not certain that a missense mutation produces a functional deficit. Functional assays of BRCA2 missense mutations are not readily available, but these new structural data may allow modeling and interpretation of how a mutation affects critical interaction sites. Returning to the question of how cancer arises in cells with deficiencies in BRCA2 (or BRCA1), it seems likely that the impaired function of homologous recombination is a key step. A “mutator” phenotype can be triggered by a defect in the regulation of homologous recombination, either by making the process error-prone or by shunting repair events into a nonhomologous end-joining pathway that is inherently mutagenic. Additional mutational steps, to bypass the proliferative block, are also necessary to allow a net growth in the number of cancer cells. However, what remains is the unsolved mystery of why this described defect in homologous recombination leads to the tissue-specific cancers of the breast and ovary. Proliferation in breast or ovarian epithelium may be associated with higher levels of endogenous DNA damage, relative to other tissues, which leads to replication stalling and requires homologous recombination for replication restart. Research into BRCA1 and BRCA2 function is now at a fever pitch: the next few years should be productive times in dissecting the function of these interesting large proteins. Simon N. Powell, Henning Willers, and Fen Xia Department of Radiation Oncology Massachusetts General Hospital and Harvard Medical School Building 149, 13th Street Charlestown, Massachusetts 02129 Selected Reading
allowing the formation of a Rad51 filament; alternatively, BRCA2 could primarily bind ssDNA and directly facilitate the formation of Rad51 filaments. The role of the “tower” region of the C terminus of BRCA2, with the recombinase motif, is more difficult to rationalize with the displacement and loading model, unless the loading of Rad51, filament formation, and strand exchange are interlinked processes, with BRCA2 being the linking protein (see Figure). This model was partly suggested in a recent editorial (Wilson and Elledge, 2002), in which BRCA2 was proposed to facilitate rate of loading of Rad51 and the organization of the Rad51 filament. The recent structural data strongly support the view that the primary role of BRCA2 is to facilitate the mechanism of homologous recombination. Prior suggestions
Davies, A.A., Masson, J.Y., McIlwraith, M.J., Stasiak, A.Z., Stasiak, A., Venkitaraman, A.R., and West, S.C. (2001). Mol. Cell 7, 273–282. Moynahan, M.E., Pierce, A.J., and Jasin, M. (2001). Mol. Cell 7, 263–272. Pellegrini, L., Yu, D.S., Lo, T., Anand, S., Lee, M., Blundell, T.L., and Venkitaraman, A.R. (2002). Nature 420, 287–293. Tutt, A., Bertwistle, D., Valentine, J., Gabriel, A., Swift, S., Ross, G., Griffin, C., Thacker, J., and Ashworth, A. (2001). EMBO J. 20, 4704–4716. Wilson, J.H., and Elledge, S.J. (2002). Science 297, 1822–1823. Xia, F., Taghian, D.G., DeFrank, J.S., Zeng, Z.C., Willers, H., Iliakis, G., and Powell, S.N. (2001). Proc. Natl. Acad. Sci. USA 98, 8644– 8649. Yang, H., Jeffrey, P.D., Miller, J., Kinnucan, E., Sun, Y., Thoma, N.H., Zheng, N., Chen, P.L., Lee, W.H., and Pavletich, N.P. (2002). Science 297, 1837–1848.