BRCA1 and DNA damage response pathways

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Read about BRCA1 localization, phosphorylation, and the role BRCA1 plays in double strand break repair. 

By Amanda Simons, Ph.D (Harvard)

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​Contents

• BRCA1 localization
• BRCA1 phosphorylation
• BRCA1 and double strand break repair
• BRCA1 and other forms of DNA damage repair
• References​ 

BRCA1 localization

BRCA1 is a breast and ovarian specific tumor suppressor. Approximately 50% of hereditary breast cancers can be attributed to BRCA1, and women carrying heterozygous mutations in BRCA1 have approximately a 90% lifetime risk of developing cancer. 
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BRCA1 is a 220-kd primarily nuclear phosphoprotein. Expression of BRCA1 varies with the cell cycle, reaching highest expression in S phase ³, during which time it forms nuclear structures visible by immunofluorescence as punctate nuclear foci. Following DNA damage, BRCA1 is dispersed from the S-phase foci and relocalized to damage-induced foci. BRCA1 colocalizes in these damage-induced foci with a number of proteins involved in the DNA damage response¹⁶ . The phosphorylated histone H2A.X, which marks tracts of chromatin surrounding damaged DNA, significantly overlaps with BRCA1following DNA damage; thus, BRCA1 damage-induced foci are thought to be sites of DNA repair¹³. 
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Most of the cellular pool of BRCA1 exists as a heterodimer with BARD1, suggesting that the functions of the two proteins are tightly linked. Together, BRCA1/BARD1 form an active E3 ubiquitin ligase, and the ubiquitination function of the heterodimer likely plays a large role in BRCA1 tumor suppression. In addition, a body of evidence suggests that the BRCA1/BARD1 heterodimer may play a direct role in DNA repair. Notably, BRCA1/BARD1 interacts directly with aberrant DNA structures ^{12, 18} as well as with a number of proteins involved in the DNA damage response. 

BRCA1 phosphorylation

BRCA1 relocalization to damage-induced foci coincides with its phosphorylation¹⁶. BRCA1is phosphorylated by ATM, ATR, and Chk2 on several serines throughout the length of the protein in response to DNA damage, suggesting that phosphorylation of BRCA1 plays a role in DNA damage response ^{4, 7, 9}. Sites of phosphorylation on BRCA1 vary with the type of DNA damage, with ATM-sites specific for ionizing radiation and other damaging agents that cause double strand breaks and ATR-sites specific for damage caused by ultraviolet radiation⁷. These phosphorylation events appear to have downstream signaling consequences, as certain damage-induced phosphorylation events elicited by ATM first require proper phosphorylation of BRCA1⁶. ​​

BRCA1 and double strand break repair

BRCA1 appears to play a role in two distinct pathways for double strand break repair, non-homologous end joining and homology-directed repair. Non-homologous end joining is an error-prone system for DNA repair that can result in loss of sequence information around the break. Homology-directed repair, in contrast, uses regions of homology to preserve sequence integrity surrounding a double-strand break. Homology directed repair might be as simple as exposing single-stranded DNA on both sides of the break through action of an exonuclease, enabling annealing of exposed complementary sequence. Homology-directed repair may alternatively be more complex, involving homologous recombination proteins in a process that requires resection of single-strands around the break, a search for homologous sequence on an intact molecule, synthesis of new DNA using a homologous sequence as a template, and resolution of the intertwined recombinant DNA strands (Figure 1a). This process results in double-strand break repair with greater fidelity than single-strand annealing or non-homologous end joining. However, the requirement for a homologous template for repair is a limitation of double-strand-break repair by homologous recombination. In mammalian cells, repair by homologous recombination is therefore primarily restricted to late S or G2 phase of the cell cycle, when an identical sister chromatid is available¹⁵.

Studies examining the role of BRCA1 in DNA repair have yielded seemingly conflicting results, with BRCA1implicated in both non-homologous end joining and homologous repair. BRCA1 interacts with proteins involved in both the non-homologous end-joining pathway (including the Mre11, Rad50, Nbs1 complex)⁸ and homologous repair (RAD51 and BRCA2, among others)^2, 17 and forms damage-induced foci irrespective of the cell cycle. In addition, a number of cell-based experiments support involvement of BRCA1 in both pathways. Studies of BRCA1 using a reporter for homology-directed repair have shown that BRCA1 promotes both single-strand annealing and homologous recombination, suggesting that BRCA1 is involved in homology-directed repair upstream of the divergence of SSA and HR (Moynahan et al., 1999; Snouwaert et al., 1999; Stark et al., 2004). On the other hand, extracts from BRCA1-deficient cells are deficient in end-joining (Zhong et al., 2002). Together, these data suggest that BRCA1 is either involved in double strand break repair upstream of both NHEJ and HDR, or BRCA1 functions in a manner common to both types of DNA repair. 

BRCA1 and other forms of DNA damage repair

BRCA1 has also been linked with a number of other DNA repair processes, including mismatch repair (through interactions with the mismatch repair proteins MSH2, MSH6, and MLH1) and inter-strand crosslink repair¹⁹. Intriguingly, there is a growing body of evidence linking BRCA1 with the Fanconi anemia pathway for inter-strand crosslink repair. Fanconi anemia, a disorder characterized by developmental defects, chromosomal abnormalities, and susceptibility to DNA cross-linking agents, is caused by recessive mutations in any one of at least twelve genes. Although BRCA1 does not appear to be a Fanconi protein, several BRCA1interactors are linked to Fanconi anemia, including BRCA2 (FancD1), BACH1 (FancJ), and FancA^{1, 2, 5}. 

Because inter-strand crosslinks may be converted to double-strand breaks in the course of repair (Figure 1b)^{10, 11, 14}, BRCA1 may affect the Fanconi pathway after a partially-repaired DNA is shunted into a homologous recombination pathway. On the other hand, because BRCA1 appears to play a role in repairing a disparate collection of DNA lesions, it is possible that BRCA1 acts in a manner that is upstream of or common to many DNA repair pathways, such as acting as a sensor for DNA damage. ​

Figure 1: Mechanisms of DNA repair

Figure legend

Figure 1a. Double-strand break repair by homologous recombination. Single stranded regions of DNA surrounding a double strand break are exposed by an exonuclease, leaving a 3’ tail. This single stranded tail is used to search for homology in an unbroken homologous chromosome and then invades the intact duplex. A double Holliday junction structure is formed, allowing the unbroken chromosome to be used as a template for DNA synthesis. The Holliday junctions can branch migrate, forming regions of heteroduplex DNA that may later result in gene conversion. Because Holliday structures are resolved in one of two orientations by cleaving opposite strands, four distinct products or resolution are possible. For simplicity, only one is shown. 

Figure 1b. Inter-strand crosslink repair may involve homologous recombination. Two models for inter-strand crosslink repair are shown. On the left, a replication-independent mechanism involves strand invasion and Holliday junction formation following unhooking of the inter-strand crosslink. On the right, replication is stalled when the fork reaches an inter-strand crosslink. Cleavage of the stalled fork results in formation of a double-strand break. Following repair of the inter-strand crosslink, strand invasion initiates the reformation of the replication fork prior to replication re-initiation. 

References

1. Cantor, S. B., Bell, D. W., Ganesan, S., Kass, E. M., Drapkin, R., Grossman, S., Wahrer, D. C., Sgroi, D. C., Lane, W. S., Haber, D. A., and Livingston, D. M. (2001). BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell 105, 149-160.

2. Chen, J., Silver, D. P., Walpita, D., Cantor, S. B., Gazdar, A. F., Tomlinson, G., Couch, F. J., Weber, B. L., Ashley, T., Livingston, D. M., and Scully, R. (1998). Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol Cell 2, 317-328.

3. Chen, Y., Farmer, A. A., Chen, C. F., Jones, D. C., Chen, P. L., and Lee, W. H. (1996). BRCA1 is a 220-kDa nuclear phosphoprotein that is expressed and phosphorylated in a cell cycle-dependent manner. Cancer Res 56, 3168-3172.

4. Cortez, D., Wang, Y., Qin, J., and Elledge, S. J. (1999). Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. Science 286, 1162-1166.

5. Folias, A., Matkovic, M., Bruun, D., Reid, S., Hejna, J., Grompe, M., D'Andrea, A., and Moses, R. (2002). BRCA1 interacts directly with the Fanconi anemia protein FANCA. Hum Mol Genet 11, 2591-2597.

6. Foray, N., Marot, D., Gabriel, A., Randrianarison, V., Carr, A. M., Perricaudet, M., Ashworth, A., and Jeggo, P. (2003). A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein. Embo J 22, 2860-2871.

7. Gatei, M., Zhou, B. B., Hobson, K., Scott, S., Young, D., and Khanna, K. K. (2001). Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo assessment using phospho-specific antibodies. J Biol Chem 276, 17276-17280.

8. Greenberg, R. A., Sobhian, B., Pathania, S., Cantor, S. B., Nakatani, Y., and Livingston, D. M. (2006). Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes. Genes Dev 20, 34-46.

9. Lee, J. S., Collins, K. M., Brown, A. L., Lee, C. H., and Chung, J. H. (2000). hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature 404, 201-204.

10. McHugh, P. J., Spanswick, V. J., and Hartley, J. A. (2001). Repair of DNA interstrand crosslinks: molecular mechanisms and clinical relevance. Lancet Oncol 2, 483-490.

11. Niedernhofer, L. J., Essers, J., Weeda, G., Beverloo, B., de Wit, J., Muijtjens, M., Odijk, H., Hoeijmakers, J. H., and Kanaar, R. (2001). The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells. Embo J 20, 6540-6549.

12. Paull, T. T., Cortez, D., Bowers, B., Elledge, S. J., and Gellert, M. (2001). Direct DNA binding by Brca1. Proc Natl Acad Sci U S A 98, 6086-6091.

13. Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. (2000). A critical role for histone H2A.X in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10, 886-895.

14. Rothfuss, A., and Grompe, M. (2004). Repair kinetics of genomic interstrand DNA cross-links: evidence for DNA double-strand break-dependent activation of the Fanconi anemia/BRCA pathway. Mol Cell Biol 24, 123-134.

15. Rothkamm, K., Kruger, I., Thompson, L. H., and Lobrich, M. (2003). Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol Cell Biol 23, 5706-5715.

16. Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997a). Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90, 425-435.

17. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and Livingston, D. M. (1997b). Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell 88, 265-275.

18. Simons, A. M., Horwitz, A. A., Starita, L. M., Griffin, K., Williams, R. S., Glover, J. N., and Parvin, J. D. (2006). BRCA1 DNA-binding activity is stimulated by BARD1. Cancer Res 66, 2012-2018.

19. Wang, Y., Cortez, D., Yazdi, P., Neff, N., Elledge, S. J., and Qin, J. (2000). BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev 14, 927-939.

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