Genes are the instruction manuals of life and contain the information needed to build the proteins that keep cells alive. Over time, genes can accumulate errors or mutations and eventually become faulty, which can lead to diseases like cancer. Sometimes mutations can be passed on through generations and increase the chances of getting cancer. The BRCA1 gene, for example, provides instructions for making a protein that helps to repair or remove damaged DNA and stops cells from growing uncontrollably. When the BRCA1 gene becomes faulty, cells could continue to grow with damaged DNA. This makes it more likely for cancer to develop, especially breast cancer and ovarian cancer.

However, not all changes in BRCA1 gene cause the protein to become faulty or lead to cancer. In fact, about 30% of BRCA1 gene changes identified by genetic tests are referred to as ‘variants of uncertain clinical significance’, meaning that it is not clear if these variants are indeed mutations that could affect the clinical outcome of the people that carry them. Software predictions based largely on patient data have categorized many of these variants as not cancer-causing, but the majority still need to be experimentally tested and confirmed. Many studies have tried to determine the effect of selected variants on the BRCA1 protein, but a complete picture remains lacking.

Now, Anantha et al. have tested the top 22 common variants in the BRCA1 gene, some of which had known effects and some did not. The study tested how these variants affect the ability of the protein to repair damaged DNA and the efficacy of chemotherapies targeting cancer cells with a DNA repair defect. The experiments revealed that three specific parts of the protein must remain intact in order for the protein to carry out this activity, i.e. mutations that affect these three areas are likely to cause cancer and also make cancer cells vulnerable to these chemotherapies. Anantha et al. also generated a series of 10 artificially shortened BRCA1 proteins, each missing a specific part, to determine the possible effects of other variants in those missing parts.

Together the findings reveal previously unknown effects of certain variants that are commonly seen in cancer patients as well new insights into how the BRCA1 protein repairs DNA. The next step will be to assess rarer variants where little data is available. A better understanding of how these variants affect DNA repair and drug response will help to improve the genetic counseling and treatment of patients with breast cancer and ovarian cancer.

Germline, heterozygous mutations in BRCA1 confer high risk of breast and ovarian cancer development in an autosomal dominant fashion (Couch et al., 2014; Fackenthal and Olopade, 2007). BRCA1 has been implicated in numerous cellular processes including DNA repair, cell cycle checkpoints, centrosome duplication, and transcriptional regulation, etc. (Deng, 2006; Mullan et al., 2006; Roy et al., 2012). Ever since BRCA1 was found to localize to discrete nuclear foci and colocalize with the recombination enzyme RAD51 (Scully et al., 1997), its function in homologous recombination (HR)-based repair of DNA double strand breaks (DSBs) has been a subject of intense study (Moynahan and Jasin, 2010). Tumors arising from BRCA1 mutation carriers usually show loss of the wild-type (wt) allele, which renders tumor cells biallelically null for the gene. It is generally believed that genome instability resulting from the DNA repair defect following the loss of BRCA1 is a driver of tumor development (Li and Greenberg, 2012; Venkitaraman, 2014). Importantly, the very DNA repair defect that leads to tumor development is also an ‘Achilles’ Heel’ of the resulting tumor cells, which can be selectively killed by suitable DNA-damaging agents that target HR defect, such as platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors (Lord and Ashworth, 2016).

The human BRCA1 gene consists of 24 exons encoding a large polypeptide of 1863 amino acid residues. BRCA1 contains a RING domain at the N terminus and a tandem BRCT domain at the C terminus (Figure 1A). Two nuclear localization signals (NLSs) facilitate the localization of BRCA1 primarily to the nucleus (Chen et al., 1996), whereas a nuclear export signal (NES) can mediate its cytoplasmic export (Rodríguez and Henderson, 2000). The RING domain of BRCA1 binds to a similar domain in its close partner BARD1 (Wu et al., 1996), leading to the formation of a stoichiometric complex that possesses substantial ubiquitin E3 ligase activity (Hashizume et al., 2001; Ruffner et al., 2001). At the same time, binding of BARD1 to BRCA1 shields the NES and helps retain BRCA1 in the nucleus (Fabbro et al., 2002). The BRCT domain directly binds, in a phosphorylation-dependent manner, to at least three other proteins, namely BRIP1, CtIP and Abraxas, all of which have function in DNA repair (Huen et al., 2010; Jiang and Greenberg, 2015). Additionally, BRCA1 contains a highly conserved coiled-coil (CC) motif that directly binds PALB2, the partner and localizer of BRCA2 (Xia et al., 2006), which links BRCA1 and BRCA2 in the HR pathway (Sy et al., 2009; Zhang et al., 2009a, 2009b).

Figure 1 with 2 supplements see all

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Figure 1—source data 1

HR activities of the BRCA1 variants and mutants analzyed in this study.

See texts and materials and methods for details.

https://doi.org/10.7554/eLife.21350.004

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The Breast Cancer Information Core (BIC) database was the first major public database into which patient-derived mutations and variants were deposited. It contains a total of 15,311 entries of BRCA1 sequence alterations, among which 6133 (40%) are frameshift or protein-truncating mutations, which can generally be classified as pathogenic. Importantly, 4577 (30%) are missense variants, most of which are considered to be of ‘unknown clinical importance’, or commonly known as variants of unknown (or uncertain) clinical significance (VUSs), the interpretation of which remains a challenge (Couch et al., 2014). Recently, more data have been deposited into the ClinVar database, which now includes all BIC cases along with ones from multiple other sources. Based largely on genetic and epidemiological evidence, a large number of VUSs has been designated by ClinVar as ‘benign’. However, the majority of the variants have not been functionally characterized.

A number of studies have been conducted to assess the functions of select BRCA1 VUS in transcription, DNA repair, and in supporting the viability of mouse embryonic stem (ES) cells (Bouwman et al., 2013; Chang et al., 2009; Millot et al., 2012; Ransburgh et al., 2010; Towler et al., 2013). However, data on the impact of VUS on drug sensitivity have only begun to emerge. In particular, a recent study used a recombinase-mediated cassette exchange (RMCE) approach to profile 74 patient-derived missense variants for their activities in conferring resistance to cisplatin and a PARP inhibitor in mouse ES cells, and it found that all functionally deleterious variants were confined to the RING and BRCT domains (Bouwman et al., 2013).

In this study, we systematically assessed HR and SSA activities of 32 natural variants, including 11-patient-derived missense mutations that affect known protein-protein interactions and the top 22-patient-associated missense variants, as well as 10 overlapping deletion mutations that span the entire length of the protein. Our data clearly demonstrate that the HR function of BRCA1 requires the RING, CC and BRCT domains. Interestingly, deletion of residues 1056–1357 (BD7) caused ~50% reduction in both HR and SSA activity without affecting the binding of any of the above known partners. This segment of BRCA1 does not contain any recognizable domains and has not been implicated in HR before, suggesting the existence of a novel binding partner or regulatory mechanism. Together, these results establish a functional landscape of BRCA1 for its HDR function.

The RING domain of BRCA1 binds to the RING domain of BARD1, and the BRCA1/BARD1 heterodimer formation is critical for their stability, their E3 ubiquitin ligase activity and the nuclear retention of BRCA1 (Fabbro et al., 2002; Hashizume et al., 2001; Ruffner et al., 2001). Conditional knock out of either Brca1 or Bard1 in murine mammary epithelial cells led to the development of mammary carcinomas that are indistinguishable from each other (Shakya et al., 2008), and the BRCA1 C61G mutant that abrogates BARD1 binding failed to suppress mammary tumor development in mice (Drost et al., 2011). Moreover, the I26A mutation that abolishes its E3 ligase activity did not affect HR activity or mitomycin C (MMC) sensitivity (Reid et al., 2008), nor did it affect tumor suppression in mice (Shakya et al., 2011). These findings suggested that the BARD1-binding function of the RING domain, rather than its E3 ligase activity, plays a key role in BRCA1 HR and tumor suppression activity. In this regard, our findings on the I26A mutation are worth noting. First, it caused a significant impairment in BARD1 binding (Figure 5A); second, it moderately reduced HR efficiency (Figure 5B); and third, it caused sensitivity to both cisplatin and olaparib under the condition used (Figure 5C). The reduced BARD1 binding is consistent with the fact that I26A mutant mouse cells had substantially reduced amount of both BRCA1 and BARD1 (Reid et al., 2008). Also, a recent report showed that HeLa cells expressing only BRCA1-I26A were similarly sensitive to olaparib as BRCA1 knockdown cells (Densham et al., 2016). Thus, it is safe to conclude that the I26A mutation affects HR and drug resistance in human cells. The discrepancy in observed HR activities and drug sensitivities in mouse ES cells and human cancer cells may be explained by the different tolerance for partial loss of BARD1 binding in the two systems or the presence of other mutations in cancer cells. At the same time, although I26A mutant mouse cells showed no defect in apparent HR activity or MMC resistance, reduced gene targeting efficiency and increased chromosomal abnormalities after MMC treatment were noted (Reid et al., 2008), indicative of a possible defect in HR in mouse cells as well. Despite the discrepancy, it should be noted that our results do not contradict the argument that the E3 ligase activity of BRCA1 is dispensable for tumor suppression (Shakya et al., 2011).

Mutations of the BRCT domain dramatically abolish HR activity, and the mechanism is also complex. First, this domain directly interacts with at least three proteins, Abraxas, a scaffold protein (Wang et al., 2007), BRIP1, a DNA helicase (Cantor et al., 2001), and CtIP, an endonuclease involved in resection (Sartori et al., 2007). It has recently been shown that CtIP binding to BRCA1 is dispensable for CtIP-mediated DNA resection (Polato et al., 2014; Reczek et al., 2013), yet to what extent BRCA1 HR activity depends on Abraxas and BRIP1 remains unclear. Second, mutation of the BRCT domain causes a major defect in the nuclear accumulation of BRCA1 (Figure 4A–B), supporting a key role of the BRCT domain for BRCA1 nuclear entry. Our finding that BARD1 can significantly rescue nuclear localization of BRCT mutants but not their HR defects (Figure 4F) also supports an important and direct role of the BRCT domain for BRCA1 DNA repair activity. Collectively, our data demonstrate the dual role of the BRCT domain and underscore a complex regulation of BRCA1 localization and repair function by the RING and BRCT domains and their binding partners (Figure 7A).

Figure 7

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Consistent with early observations made in mouse cells (Stark et al., 2004), BRCA1 depletion reduced the efficiency of both HR and SSA, whereas depletion of BRCA2 led to severe loss of HR (data not shown) but increased SSA (Figure 2B). As efficient resection is necessary for both repair mechanisms, the data is consistent with the notion that BRCA1 plays a key role in resection (Bunting et al., 2010). Importantly, we found that depletion of PALB2 showed similar, if not more dramatic, induction of SSA as did BRCA2 depletion (Figure 2B). Moreover, all three point mutations in BRCA1 that affect PALB2 binding led to increased SSA usage (Figure 2A), and two different PALB2 mutants with greatly impaired BRCA1-binding capacity both failed to suppress the upregulation of SSA in PALB2-depleted cells (Figure 2F). These results clearly demonstrate that the direct interaction between BRCA1 and PALB2 is required for suppressing, or at least preventing, SSA, while promoting HR (Figure 7A–B). As such, our results support two critical roles of BRCA1 function in DSB repair, promoting resection and recruiting/stimulating PALB2, which then channels the ssDNA down the HR path (Figure 7B). Based on the available data, PALB2 is the key partner of BRCA1 specifically for its HR function and the primary switch point, acting upstream of BRCA2, for HDR pathways following resection.

VUSs are commonly found during clinical genetics tests; however, their biological and clinical significance is often difficult to ascribe with confidence, and this uncertainty poses significant challenges for both clinicians and patients. Our results demonstrate that relatively common VUSs outside the RING and BRCT domains do not significantly affect the HDR activity of BRCA1. Our findings are mostly consistent with several previous studies (Table 2), including the above noted mouse ES cell-based study that covered 6 of the top 22 variants (Bouwman et al., 2013); on the rare PALB2-binding mutants L1407P and M1411T, however, our results showed that they are severely defective in HR (Figure 1D) and conferring drug resistance (Figure 3C), whereas their effects in mouse cells were less pronounced. Moreover, M1400V, which was found to be neutral in the mouse cell system, shows clear sensitivity to olaparib treatment in the human cell line. This discrepancy could again be due to a difference in the thresholds of human and mouse cells to tolerate partial loss of BRCA1-PALB2 interaction or the presence of other mutations in the human cancer cells.

Platinum-based therapeutics have been commonly used for treatment of ovarian cancer, and olaparib has recently been approved for the treatment of BRCA1/2 mutant ovarian cancer, with multiple trials on breast and other cancers ongoing or recently completed. These agents generate lesions that block or collapse DNA replication forks, leading to DSBs that require HR and therefore BRCA1/2 for repair (Lord and Ashworth, 2016). Under the condition used, olaparib appeared to have a better selectivity in killing MDA-MB-436 cells expressing mutant BRCA1 proteins. Although this could be due to a slightly lower than optimal concentration of cisplatin used, the possibility that cisplatin is intrinsically less selective than olaparib cannot be ruled out. Based on data from this study and other recent studies, patients with relatively common missense mutations in the RING and BRCT domains, such as C61G and A1708E, respectively, or rare mutations in the CC motif, such as L1407P or M1411T, are likely to respond to platinum- or PARPi-based therapies, unless resistance mechanisms have already occurred prior to treatment or are induced by the treatment (Lord and Ashworth, 2013). However, except for C61G and A1708E, all other top 22 missense variants are fully or mostly functional both in HR and in conferring cisplatin and olaparib resistance (Figure 3C), which corroborates with clinical genetics evidence to further cast doubts on their pathogenicity and diminish the likelihood of a positive response of the patients to the above regimens. Based on the results of our deletion analyses and other available data, it is unlikely for any VUS outside the RING, CC and BRCT domains to be strongly pathogenic or to elicit high sensitivity to platinum or PARPi therapies (unless it destabilizes the transcript or protein). However, the region deleted in BD7 (residues 1056–1,307) may harbor VUS that have intermediate effects.

1. 1. Bouwman P
   2. van der Gulden H
   3. van der Heijden I
   4. Drost R
   5. Klijn CN
   6. Prasetyanti P
   7. Pieterse M
   8. Wientjens E
   9. Seibler J
   10. Hogervorst FB
   11. Jonkers J

   (2013) A high-throughput functional complementation assay for classification of BRCA1 missense variants

   Cancer Discovery 3:1142–1155.

   https://doi.org/10.1158/2159-8290.CD-13-0094

   • PubMed
   • Google Scholar
2. 1. Brzovic PS
   2. Keeffe JR
   3. Nishikawa H
   4. Miyamoto K
   5. Fox D
   6. Fukuda M
   7. Ohta T
   8. Klevit R

   (2003) Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex

   PNAS 100:5646–5651.

   https://doi.org/10.1073/pnas.0836054100

   • PubMed
   • Google Scholar
3. 1. Bunting SF
   2. Callén E
   3. Wong N
   4. Chen HT
   5. Polato F
   6. Gunn A
   7. Bothmer A
   8. Feldhahn N
   9. Fernandez-Capetillo O
   10. Cao L
   11. Xu X
   12. Deng CX
   13. Finkel T
   14. Nussenzweig M
   15. Stark JM
   16. Nussenzweig A

   (2010) 53bp1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks

   Cell 141:243–254.

   https://doi.org/10.1016/j.cell.2010.03.012

   • PubMed
   • Google Scholar
4. 1. Cantor SB
   2. Bell DW
   3. Ganesan S
   4. Kass EM
   5. Drapkin R
   6. Grossman S
   7. Wahrer DC
   8. Sgroi DC
   9. Lane WS
   10. Haber DA
   11. Livingston DM

   (2001) BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function

   Cell 105:149–160.

   https://doi.org/10.1016/S0092-8674(01)00304-X

   • PubMed
   • Google Scholar
5. 1. Chang S
   2. Biswas K
   3. Martin BK
   4. Stauffer S
   5. Sharan SK

   (2009) Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations

   Journal of Clinical Investigation 119:3160–3171.

   https://doi.org/10.1172/JCI39836

   • PubMed
   • Google Scholar
6. 1. Chen CF
   2. Li S
   3. Chen Y
   4. Chen PL
   5. Sharp ZD
   6. Lee WH

   (1996) The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor

   Journal of Biological Chemistry 271:32863–32868.

   https://doi.org/10.1074/jbc.271.51.32863

   • PubMed
   • Google Scholar
7. 1. Chen J
   2. Silver DP
   3. Walpita D
   4. Cantor SB
   5. Gazdar AF
   6. Tomlinson G
   7. Couch FJ
   8. Weber BL
   9. Ashley T
   10. Livingston DM
   11. Scully R

   (1998) Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells

   Molecular Cell 2:317–328.

   https://doi.org/10.1016/S1097-2765(00)80276-2

   • PubMed
   • Google Scholar
8. 1. Couch FJ
   2. Nathanson KL
   3. Offit K

   (2014) Two decades after BRCA: setting paradigms in personalized Cancer care and prevention

   Science 343:1466–1470.

   https://doi.org/10.1126/science.1251827

   • PubMed
   • Google Scholar
9. 1. Deng CX

   (2006) BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and Cancer evolution

   Nucleic Acids Research 34:1416–1426.

   https://doi.org/10.1093/nar/gkl010

   • PubMed
   • Google Scholar
10. 1. Densham RM
    2. Garvin AJ
    3. Stone HR
    4. Strachan J
    5. Baldock RA
    6. Daza-Martin M
    7. Fletcher A
    8. Blair-Reid S
    9. Beesley J
    10. Johal B
    11. Pearl LH
    12. Neely R
    13. Keep NH
    14. Watts FZ
    15. Morris JR

    (2016) Human BRCA1-BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection

    Nature Structural & Molecular Biology 23:647–655.

    https://doi.org/10.1038/nsmb.3236

    • PubMed
    • Google Scholar
11. 1. Drost R
    2. Bouwman P
    3. Rottenberg S
    4. Boon U
    5. Schut E
    6. Klarenbeek S
    7. Klijn C
    8. van der Heijden I
    9. van der Gulden H
    10. Wientjens E
    11. Pieterse M
    12. Catteau A
    13. Green P
    14. Solomon E
    15. Morris JR
    16. Jonkers J

    (2011) BRCA1 RING function is essential for tumor suppression but dispensable for therapy resistance

    Cancer Cell 20:797–809.

    https://doi.org/10.1016/j.ccr.2011.11.014

    • PubMed
    • Google Scholar
12. 1. Elstrodt F
    2. Hollestelle A
    3. Nagel JH
    4. Gorin M
    5. Wasielewski M
    6. van den Ouweland A
    7. Merajver SD
    8. Ethier SP
    9. Schutte M

    (2006) BRCA1 mutation analysis of 41 human breast Cancer cell lines reveals three new deleterious mutants

    Cancer Research 66:41–45.

    https://doi.org/10.1158/0008-5472.CAN-05-2853

    • PubMed
    • Google Scholar
13. 1. Fabbro M
    2. Rodriguez JA
    3. Baer R
    4. Henderson BR

    (2002) BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export

    Journal of Biological Chemistry 277:21315–21324.

    https://doi.org/10.1074/jbc.M200769200

    • PubMed
    • Google Scholar
14. 1. Fackenthal JD
    2. Olopade OI

    (2007) Breast Cancer risk associated with BRCA1 and BRCA2 in diverse populations

    Nature Reviews Cancer 7:937–948.

    https://doi.org/10.1038/nrc2054

    • PubMed
    • Google Scholar
15. 1. Foo TK
    2. Tischkowitz M
    3. Simhadri S
    4. Boshari T
    5. Zayed N
    6. Burke KA
    7. Berman SH
    8. Blecua P
    9. Riaz N
    10. Huo Y
    11. Ding YC
    12. Neuhausen SL
    13. Weigelt B
    14. Reis-Filho JS
    15. Foulkes WD
    16. Xia B

    (2017) Compromised BRCA1-PALB2 interaction is associated with breast Cancer risk

    Oncogene.

    https://doi.org/10.1038/onc.2017.46

    • PubMed
    • Google Scholar
16. 1. Greenberg RA
    2. Sobhian B
    3. Pathania S
    4. Cantor SB
    5. Nakatani Y
    6. Livingston DM

    (2006) Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes

    Genes & Development 20:34–46.

    https://doi.org/10.1101/gad.1381306

    • PubMed
    • Google Scholar
17. 1. Guidugli L
    2. Pankratz VS
    3. Singh N
    4. Thompson J
    5. Erding CA
    6. Engel C
    7. Schmutzler R
    8. Domchek S
    9. Nathanson K
    10. Radice P
    11. Singer C
    12. Tonin PN
    13. Lindor NM
    14. Goldgar DE
    15. Couch FJ

    (2013) A classification model for BRCA2 DNA binding domain missense variants based on homology-directed repair activity

    Cancer Research 73:265–275.

    https://doi.org/10.1158/0008-5472.CAN-12-2081

    • PubMed
    • Google Scholar
18. 1. Gunn A
    2. Stark JM

    (2012) I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks

    Methods in Molecular Biology 920:379–391.

    https://doi.org/10.1007/978-1-61779-998-3_27

    • PubMed
    • Google Scholar
19. 1. Hashizume R
    2. Fukuda M
    3. Maeda I
    4. Nishikawa H
    5. Oyake D
    6. Yabuki Y
    7. Ogata H
    8. Ohta T

    (2001) The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation

    Journal of Biological Chemistry 276:14537–14540.

    https://doi.org/10.1074/jbc.C000881200

    • PubMed
    • Google Scholar
20. 1. Huen MS
    2. Sy SM
    3. Chen J

    (2010) BRCA1 and its toolbox for the maintenance of genome integrity

    Nature Reviews Molecular Cell Biology 11:138–148.

    https://doi.org/10.1038/nrm2831

    • PubMed
    • Google Scholar
21. 1. Jensen RB
    2. Carreira A
    3. Kowalczykowski SC

    (2010) Purified human BRCA2 stimulates RAD51-mediated recombination

    Nature 467:678–683.

    https://doi.org/10.1038/nature09399

    • PubMed
    • Google Scholar
22. 1. Jiang Q
    2. Greenberg RA

    (2015) Deciphering the BRCA1 tumor Suppressor Network

    Journal of Biological Chemistry 290:17724–17732.

    https://doi.org/10.1074/jbc.R115.667931

    • PubMed
    • Google Scholar
23. 1. Li ML
    2. Greenberg RA

    (2012) Links between genome integrity and BRCA1 tumor suppression

    Trends in Biochemical Sciences 37:418–424.

    https://doi.org/10.1016/j.tibs.2012.06.007

    • PubMed
    • Google Scholar
24. 1. Liu J
    2. Doty T
    3. Gibson B
    4. Heyer WD

    (2010) Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA

    Nature Structural & Molecular Biology 17:1260–1262.

    https://doi.org/10.1038/nsmb.1904

    • PubMed
    • Google Scholar
25. 1. Lord CJ
    2. Ashworth A

    (2013) Mechanisms of resistance to therapies targeting BRCA-mutant cancers

    Nature Medicine 19:1381–1388.

    https://doi.org/10.1038/nm.3369

    • PubMed
    • Google Scholar
26. 1. Lord CJ
    2. Ashworth A

    (2016) BRCAness revisited

    Nature Reviews Cancer 16:110–120.

    https://doi.org/10.1038/nrc.2015.21

    • PubMed
    • Google Scholar
27. 1. Lu C
    2. Xie M
    3. Wendl MC
    4. Wang J
    5. McLellan MD
    6. Leiserson MD
    7. Huang KL
    8. Wyczalkowski MA
    9. Jayasinghe R
    10. Banerjee T
    11. Ning J
    12. Tripathi P
    13. Zhang Q
    14. Niu B
    15. Ye K
    16. Schmidt HK
    17. Fulton RS
    18. McMichael JF
    19. Batra P
    20. Kandoth C
    21. Bharadwaj M
    22. Koboldt DC
    23. Miller CA
    24. Kanchi KL
    25. Eldred JM
    26. Larson DE
    27. Welch JS
    28. You M
    29. Ozenberger BA
    30. Govindan R
    31. Walter MJ
    32. Ellis MJ
    33. Mardis ER
    34. Graubert TA
    35. Dipersio JF
    36. Ley TJ
    37. Wilson RK
    38. Goodfellow PJ
    39. Raphael BJ
    40. Chen F
    41. Johnson KJ
    42. Parvin JD
    43. Ding L

    (2015) Patterns and functional implications of rare germline variants across 12 cancer types

    Nature Communications 6:10086.

    https://doi.org/10.1038/ncomms10086

    • PubMed
    • Google Scholar
28. 1. Ma J
    2. Cai H
    3. Wu T
    4. Sobhian B
    5. Huo Y
    6. Alcivar A
    7. Mehta M
    8. Cheung KL
    9. Ganesan S
    10. Kong AN
    11. Zhang DD
    12. Xia B

    (2012) PALB2 interacts with KEAP1 to promote NRF2 nuclear accumulation and function

    Molecular and Cellular Biology 32:1506–1517.

    https://doi.org/10.1128/MCB.06271-11

    • PubMed
    • Google Scholar
29. 1. Millot GA
    2. Carvalho MA
    3. Caputo SM
    4. Vreeswijk MP
    5. Brown MA
    6. Webb M
    7. Rouleau E
    8. Neuhausen SL
    9. Hansen T
    10. Galli A
    11. Brandão RD
    12. Blok MJ
    13. Velkova A
    14. Couch FJ
    15. Monteiro AN
    16. ENIGMA Consortium Functional Assay Working Group

    (2012) A guide for functional analysis of BRCA1 variants of uncertain significance

    Human Mutation 33:1526–1537.

    https://doi.org/10.1002/humu.22150

    • PubMed
    • Google Scholar
30. 1. Morris JR
    2. Boutell C
    3. Keppler M
    4. Densham R
    5. Weekes D
    6. Alamshah A
    7. Butler L
    8. Galanty Y
    9. Pangon L
    10. Kiuchi T
    11. Ng T
    12. Solomon E

    (2009) The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress

    Nature 462:886–890.

    https://doi.org/10.1038/nature08593

    • PubMed
    • Google Scholar
31. 1. Mortensen UH
    2. Bendixen C
    3. Sunjevaric I
    4. Rothstein R

    (1996) DNA strand annealing is promoted by the yeast Rad52 protein

    PNAS 93:10729–10734.

    https://doi.org/10.1073/pnas.93.20.10729

    • PubMed
    • Google Scholar
32. 1. Moynahan ME
    2. Jasin M

    (2010) Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis

    Nature Reviews Molecular Cell Biology 11:196–207.

    https://doi.org/10.1038/nrm2851

    • PubMed
    • Google Scholar
33. 1. Mullan PB
    2. Quinn JE
    3. Harkin DP

    (2006) The role of BRCA1 in transcriptional regulation and cell cycle control

    Oncogene 25:5854–5863.

    https://doi.org/10.1038/sj.onc.1209872

    • PubMed
    • Google Scholar
34. 1. Nakanishi K
    2. Yang YG
    3. Pierce AJ
    4. Taniguchi T
    5. Digweed M
    6. D'Andrea AD
    7. Wang ZQ
    8. Jasin M

    (2005) Human fanconi Anemia monoubiquitination pathway promotes homologous DNA repair

    PNAS 102:1110–1115.

    https://doi.org/10.1073/pnas.0407796102

    • PubMed
    • Google Scholar
35. 1. Obermeier K
    2. Sachsenweger J
    3. Friedl TW
    4. Pospiech H
    5. Winqvist R
    6. Wiesmüller L

    (2016) Heterozygous PALB2 c.1592delt mutation channels DNA double-strand break repair into error-prone pathways in breast Cancer patients

    Oncogene 35:3796–3806.

    https://doi.org/10.1038/onc.2015.448

    • PubMed
    • Google Scholar
36. 1. Oliver AW
    2. Swift S
    3. Lord CJ
    4. Ashworth A
    5. Pearl LH

    (2009) Structural basis for recruitment of BRCA2 by PALB2

    EMBO Reports 10:990–996.

    https://doi.org/10.1038/embor.2009.126

    • PubMed
    • Google Scholar
37. 1. Polato F
    2. Callen E
    3. Wong N
    4. Faryabi R
    5. Bunting S
    6. Chen HT
    7. Kozak M
    8. Kruhlak MJ
    9. Reczek CR
    10. Lee WH
    11. Ludwig T
    12. Baer R
    13. Feigenbaum L
    14. Jackson S
    15. Nussenzweig A

    (2014) CtIP-mediated resection is essential for viability and can operate independently of BRCA1

    The Journal of Experimental Medicine 211:1027–1036.

    https://doi.org/10.1084/jem.20131939

    • PubMed
    • Google Scholar
38. 1. Ransburgh DJ
    2. Chiba N
    3. Ishioka C
    4. Toland AE
    5. Parvin JD

    (2010) Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination

    Cancer Research 70:988–995.

    https://doi.org/10.1158/0008-5472.CAN-09-2850

    • PubMed
    • Google Scholar
39. 1. Reczek CR
    2. Szabolcs M
    3. Stark JM
    4. Ludwig T
    5. Baer R

    (2013) The interaction between CtIP and BRCA1 is not essential for resection-mediated DNA repair or tumor suppression

    The Journal of Cell Biology 201:693–707.

    https://doi.org/10.1083/jcb.201302145

    • PubMed
    • Google Scholar
40. 1. Reid LJ
    2. Shakya R
    3. Modi AP
    4. Lokshin M
    5. Cheng JT
    6. Jasin M
    7. Baer R
    8. Ludwig T

    (2008) E3 ligase activity of BRCA1 is not essential for mammalian cell viability or homology-directed repair of double-strand DNA breaks

    PNAS 105:20876–20881.

    https://doi.org/10.1073/pnas.0811203106

    • PubMed
    • Google Scholar
41. 1. Rodriguez JA
    2. Au WW
    3. Henderson BR

    (2004) Cytoplasmic mislocalization of BRCA1 caused by cancer-associated mutations in the BRCT domain

    Experimental Cell Research 293:14–21.

    https://doi.org/10.1016/j.yexcr.2003.09.027

    • PubMed
    • Google Scholar
42. 1. Rodríguez JA
    2. Henderson BR

    (2000) Identification of a functional nuclear export sequence in BRCA1

    Journal of Biological Chemistry 275:38589–38596.

    https://doi.org/10.1074/jbc.M003851200

    • PubMed
    • Google Scholar
43. 1. Roy R
    2. Chun J
    3. Powell SN

    (2012) BRCA1 and BRCA2: different roles in a common pathway of genome protection

    Nature Reviews Cancer 12:68–78.

    https://doi.org/10.1038/nrc3181

    • Google Scholar
44. 1. Ruffner H
    2. Joazeiro CA
    3. Hemmati D
    4. Hunter T
    5. Verma IM

    (2001) Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity

    PNAS 98:5134–5139.

    https://doi.org/10.1073/pnas.081068398

    • PubMed
    • Google Scholar
45. 1. Sartori AA
    2. Lukas C
    3. Coates J
    4. Mistrik M
    5. Fu S
    6. Bartek J
    7. Baer R
    8. Lukas J
    9. Jackson SP

    (2007) Human CtIP promotes DNA end resection

    Nature 450:509–514.

    https://doi.org/10.1038/nature06337

    • PubMed
    • Google Scholar
46. 1. Scully R
    2. Chen J
    3. Plug A
    4. Xiao Y
    5. Weaver D
    6. Feunteun J
    7. Ashley T
    8. Livingston DM

    (1997) Association of BRCA1 with Rad51 in mitotic and meiotic cells

    Cell 88:265–275.

    https://doi.org/10.1016/S0092-8674(00)81847-4

    • PubMed
    • Google Scholar
47. 1. Shakya R
    2. Reid LJ
    3. Reczek CR
    4. Cole F
    5. Egli D
    6. Lin CS
    7. deRooij DG
    8. Hirsch S
    9. Ravi K
    10. Hicks JB
    11. Szabolcs M
    12. Jasin M
    13. Baer R
    14. Ludwig T

    (2011) BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity

    Science 334:525–528.

    https://doi.org/10.1126/science.1209909

    • PubMed
    • Google Scholar
48. 1. Shakya R
    2. Szabolcs M
    3. McCarthy E
    4. Ospina E
    5. Basso K
    6. Nandula S
    7. Murty V
    8. Baer R
    9. Ludwig T

    (2008) The basal-like mammary carcinomas induced by Brca1 or Bard1 inactivation implicate the BRCA1/BARD1 heterodimer in tumor suppression

    PNAS 105:7040–7045.

    https://doi.org/10.1073/pnas.0711032105

    • PubMed
    • Google Scholar
49. 1. Stark JM
    2. Pierce AJ
    3. Oh J
    4. Pastink A
    5. Jasin M

    (2004) Genetic steps of mammalian homologous repair with distinct mutagenic consequences

    Molecular and Cellular Biology 24:9305–9316.

    https://doi.org/10.1128/MCB.24.21.9305-9316.2004

    • PubMed
    • Google Scholar
50. 1. Sy SM
    2. Huen MS
    3. Chen J

    (2009) PALB2 is an integral component of the BRCA complex required for homologous recombination repair

    PNAS 106:7155–7160.

    https://doi.org/10.1073/pnas.0811159106

    • PubMed
    • Google Scholar
51. 1. Towler WI
    2. Zhang J
    3. Ransburgh DJ
    4. Toland AE
    5. Ishioka C
    6. Chiba N
    7. Parvin JD

    (2013) Analysis of BRCA1 variants in double-strand break repair by homologous recombination and single-strand annealing

    Human Mutation 34:439–445.

    https://doi.org/10.1002/humu.22251

    • PubMed
    • Google Scholar
52. 1. Tutt A
    2. Bertwistle D
    3. Valentine J
    4. Gabriel A
    5. Swift S
    6. Ross G
    7. Griffin C
    8. Thacker J
    9. Ashworth A

    (2001) Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences

    The EMBO Journal 20:4704–4716.

    https://doi.org/10.1093/emboj/20.17.4704

    • PubMed
    • Google Scholar
53. 1. Venkitaraman AR

    (2014) Cancer suppression by the chromosome custodians, BRCA1 and BRCA2

    Science 343:1470–1475.

    https://doi.org/10.1126/science.1252230

    • PubMed
    • Google Scholar
54. 1. Wang B
    2. Matsuoka S
    3. Ballif BA
    4. Zhang D
    5. Smogorzewska A
    6. Gygi SP
    7. Elledge SJ

    (2007) Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response

    Science 316:1194–1198.

    https://doi.org/10.1126/science.1139476

    • PubMed
    • Google Scholar
55. 1. Wu LC
    2. Wang ZW
    3. Tsan JT
    4. Spillman MA
    5. Phung A
    6. Xu XL
    7. Yang MC
    8. Hwang LY
    9. Bowcock AM
    10. Baer R

    (1996) Identification of a RING protein that can interact in vivo with the BRCA1 gene product

    Nature Genetics 14:430–440.

    https://doi.org/10.1038/ng1296-430

    • PubMed
    • Google Scholar
56. 1. Xia B
    2. Sheng Q
    3. Nakanishi K
    4. Ohashi A
    5. Wu J
    6. Christ N
    7. Liu X
    8. Jasin M
    9. Couch FJ
    10. Livingston DM

    (2006) Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2

    Molecular Cell 22:719–729.

    https://doi.org/10.1016/j.molcel.2006.05.022

    • PubMed
    • Google Scholar
57. 1. Zhang F
    2. Fan Q
    3. Ren K
    4. Andreassen PR

    (2009a) PALB2 functionally connects the breast Cancer susceptibility proteins BRCA1 and BRCA2

    Molecular Cancer Research 7:1110–1118.

    https://doi.org/10.1158/1541-7786.MCR-09-0123

    • PubMed
    • Google Scholar
58. 1. Zhang F
    2. Ma J
    3. Wu J
    4. Ye L
    5. Cai H
    6. Xia B
    7. Yu X

    (2009b) PALB2 links BRCA1 and BRCA2 in the DNA-damage response

    Current Biology 19:524–529.

    https://doi.org/10.1016/j.cub.2009.02.018

    • PubMed
    • Google Scholar

Author details

1. Rachel W Anantha

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   RWA, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Srilatha Simhadri and Tzeh Keong Foo

   Competing interests

   The authors declare that no competing interests exist.

2. Srilatha Simhadri

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States
   3. Department of Medicine, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   SS, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Rachel W Anantha and Tzeh Keong Foo

   Competing interests

   The authors declare that no competing interests exist.

3. Tzeh Keong Foo

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   TKF, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Rachel W Anantha and Srilatha Simhadri

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-0168-7054

4. Susanna Miao

   Department of Genetics, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, United States

   Contribution

   SM, Data curation, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

5. Jingmei Liu

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   JL, Resources, Investigation

   Competing interests

   The authors declare that no competing interests exist.

6. Zhiyuan Shen

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   ZS, Resources, Funding acquisition, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-2834-0309

7. Shridar Ganesan

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States
   3. Department of Medicine, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   SG, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

8. Bing Xia

   Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   BX, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   xiabi@cinj.rutgers.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-3259-6139

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