J Mol Med (2003) 81:700–707 DOI 10.1007/s00109-003-0477-0
INVITED REVIEW
Anne-Renee Hartman · James M. Ford
BRCA1 and p53: compensatory roles in DNA repair
Received: 30 April 2003 / Accepted: 8 July 2003 / Published online: 9 September 2003 © Springer-Verlag 2003
Abstract The BRCA1 breast cancer susceptibility gene has been implicated in many cellular processes, yet its specific mechanism of tumor suppression remains unclear. BRCA1 plays a role in several DNA repair pathways including nucleotide excision repair (NER). Loss of the p53 tumor suppressor gene, a key regulator of NER, is an important and necessary event in the pathogenesis of BRCA1-mutated tumors. Here we discuss the role of BRCA1 and NER in breast cancer and the interactions of BRCA1 with p53 in breast tumorigenesis and suggest approaches for risk assessment and chemotherapeutic management of BRCA1-related breast cancer. ANNE RENEE HARTMAN received her training in internal medicine at The University of Chicago and is presently completing a clinical and research fellowship in medical oncology and genetics at Stanford University School of Medicine. Her research interests include the pathogenesis of inherited breast cancers and the development of early detection and prevention strategies for high-risk patients.
JAMES M. FORD received his M.D. degree from Yale School of Medicine, completed clinical training in internal medicine and medical oncology at Stanford University Medical School and a research fellowship in the Department of Biological Sciences at Stanford. He is currently the Director of the Program for Applied Cancer Genetics at Stanford and holds appointments in the Departments of Medicine (Oncology), Genetics, and Pediatrics (Medical Genetics). His laboratory research interests focus on the role of tumor suppressor genes in the regulation of DNA repair and genomic stability.
A.-R. Hartman · J. M. Ford (✉) Departments of Medicine (Oncology) and Genetics, School of Medicine, Stanford University, 269 Campus Drive, Stanford, CA, 94305, USA e-mail:
[email protected] Fax: +1-650-7251420 Present address: A.-R. Hartman, Dana Farber Cancer Institute, 44 Binney Street, Boston, MA, 02115, USA
Keywords BRCA1 · p53 · Nucleotide excision repair · Breast cancer Abbreviations BPDE: Benzo[a]pyrene-7,8-diol9,10-epoxide · ER: Estrogen receptor · FA: Fanconi anemia · GGR: Global genomic repair · IR: Ionizing radiation · NER: Nucleotide excision repair · TCR: Transcription-coupled repair · UV: Ultraviolet · XP: Xeroderma pigmentosum
BRCA1, p53, and breast cancer Inheritance of a mutation in the BRCA1 gene confers a 50–85% lifetime risk for women of developing breast cancer and a 15–45% lifetime risk of developing ovarian cancer [1, 2]. Breast cancers that develop in women carrying a BRCA1 germ-line mutation are more aggressive and confer a worse overall survival than breast cancers that occur sporadically [3, 4]. Gene expression analysis on a limited number of BRCA1- and BRCA2-associated breast tumors using DNA microarrays suggests that each has a unique profile compared to sporadic tumors, implying that these tumors undergo different genetic events in their development [5]. BRCA1-associated breast cancers have a characteristic phenotype; in general these tumors have a high mitotic index, contain p53 mutations, and do not express estrogen or progesterone receptors
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[6]. Mutations in BRCA1 are uncommon in sporadic breast cancers; however, recent studies have suggested that up to 30% of sporadic breast cancers exhibit decreased BRCA1 expression due to promoter hypermethylation [7, 8, 9, 10]. Mutations in the p53 tumor suppressor gene are found in 70–80% of breast cancers that occur in women who carry a BRCA1 mutation but only 30% of BRCA1 wildtype breast tumors, implying that loss of p53 function is an important and necessary event in the pathogenesis of BRCA1-mutated tumors [6, 11, 12]. Experiments in mice have shown that homozygous inactivation of brca1D11/D11 results in embryonic lethality that is partially rescued by inactivation of p53, suggesting that loss of p53 in brca1-deficient cells is necessary for cell viability and reflects selective pressure for loss of p53 in BRCA1-associated human breast cancers [13, 14]. After DNA damage, through exposure to doxorubicin, ionizing radiation (IR), ultraviolet (UV) irradiation or mitomycin C, protein expression of BRCA1 is downregulated in p53 wildtype cells. In contrast, protein expression of BRCA1 is stabilized or upregulated after DNA damage in both human and mouse p53 deficient cells, suggesting that the cellular consequence of loss of functional p53 is compensated for by stabilization of BRCA1 [15, 16]. Conditional knockout of brca1 in mouse mammary epithelium generate breast tumors in 25% of mice [17, 18]. The additional loss of p53 results in the development of breast tumors in 50% of these mice [17, 18]. Taken together, these data strongly suggest that loss of Fig. 1 Human nucleotide excision repair. DNA adducts are recognized by the XPE and XPC protein complexes that are transcriptionally regulated by the p53 and BRCA1 gene products. These complexes recruit the XPA/RPA complex and the larger TFIIH protein complex. The TFIIH complex contains helicases which unwind the DNA and allow the other XP proteins access for incision and excision of the damaged DNA. After excision, repair replication and ligation of the newly synthesized DNA sequence occurs
p53 is critical for the development of BRCA1-associated breast cancers.
Nucleotide excision repair and breast cancer Nucleotide excision repair (NER) is the DNA repair pathway that removes DNA adducts formed by UV irradiation and carcinogens including polyaromatic hydrocarbons, tobacco (benzo[a]pyrene-7,8-diol-9,10-epoxide, BPDE), and endogenous carcinogens, all of which may play a role in the pathogenesis of breast cancer [19, 20, 21, 22]. NER can be subdivided into two genetically distinct pathways; global genomic repair (GGR) that targets and removes lesions from the whole genome and transcription-coupled repair (TCR) that preferentially removes lesions from the transcribed strand of expressed genes [23]. The sequential steps for NER include: (a) recognition of the damaged site, (b) incision of the damaged DNA strand on each side of the defect, (c) removal of a stretch of the affected strand containing the lesion (27 to 29 nucleotides long in humans), (d) repair replication using the complementary strand as a template to replace the excised region with a corresponding stretch of normal nucleotides, and (e) ligation to join the repair patch at its 3¢ end to the contiguous parental DNA strand [23, 24] (Fig. 1). The majority of human NER genes have been cloned, and many have been shown to be mutated in inherited human NER-deficiency diseases, such as xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodys-
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breast tumors [26]. Sensitivity to BPDE may contribute to the risk of developing breast cancer [27]. Polymorphisms in the XPD and XPG genes are associated with higher levels of polycyclic aromatic hydrocarbons and cyclobutane pyrimidine dimers in DNA from breast cancer samples than in normal controls [28, 29, 30]. Loss of heterozygosity of XP genes have also been seen in breast cancer and other solid tumors [31]. Therefore the inability to repair DNA adducts due to a defect in NER may play a role in the development of breast cancer.
p53 and nucleotide excision repair Fig. 2 Defective NER, strand bias and transitional mutations. When NER is defective, the DNA polymerase often inserts adenosines opposite the covalent pyrimidine dimer during replication as a default mechanism. This results in thymidines in the newly formed strand. The consequence of this defective pathway are CCÆ TT, tandem transition mutations that are markers for NER deficiencies when identified in mutated sequences found in cancer
trophy [22]. XP is an autosomal recessive genetic disorder characterized by cutaneous photosensitivity and skin cancers with a decreased ability to repair UV-induced photoproducts. Eighty percent of XP cases are caused by a mutation in one of the seven complementation groups of the XP genes that are involved in recognizing the damaged DNA and excision of the adduct. A defective NER pathway in genetic disorders such as XP results in mutagenesis and subsequently the development of cancer in response to DNA damage. After exposure to UV a photoproduct forms between two pyrimidines on one strand of DNA. Two main types of photoproducts result from UV irradiation in the cell; the 6-4 photoproduct, which is a highly distorting lesion and repaired very quickly by the repair machinery, and the cyclobutane pyrimidine dimer, which is repaired less efficiently. In cells that have a defective NER pathway the DNA polymerase often inserts adenosines opposite the covalent pyrimidine dimer during replication as a default mechanism, resulting in thymidines in the newly formed strand. The consequence of this defective pathway are CCÆ TT transitions, tandem mutations that are specific markers for NER deficiencies when identified in mutated sequences found in cancer (Fig. 2). These are seen almost exclusively in skin cancers, particularly in XP patients, and are pathognomonic for defective NER. Tandem transition mutations have also been identified in the p53 gene associated with 2 of 82 BRCA1-mutated breast cancers. Only one other CCÆ TT transition tandem mutation in a breast cancer of unknown BRCA status out of over 10,000 cases of cancer has been identified in the p53 gene [25]. These data suggest that BRCA1 deficient cells have a defect in NER [25]. Other substrates for NER induce different mutation spectra than UV photoproducts when not properly repaired and have also been seen in breast cancer. The NER substrate BPDE is more prevalent in breast cancer tissue than in benign tissue, implying a deficit in NER in
The p53 tumor suppressor gene is a key regulator of NER [32]. p53 is a critical component of the cellular response to DNA damage and has also been shown to be involved in the regulation of cell cycle checkpoints and apoptosis [33]. Loss of p53 function specifically results in deficient GGR of UV-C induced photoproducts and several carcinogens but does not affect TCR [19, 20, 34]. We have shown that p53 affects this process through transcriptional regulation of NER genes involved in the recognition of adducts in genomic DNA including p48, the protein product of the DDB2 gene defective in XP group E (XPE) cells, XPC, the gene defective in XP group C, and GADD45, a growth arrest and DNA damage inducible gene that may facilitate chromatin unwinding in regions of damaged DNA [32, 35, 36, 37, 38]. We and others have shown that p48 and XPC, GGR-specific damage recognition factors, are involved in the earliest steps of DNA damage recognition in NER, and that in response to DNA damage p53 is induced and activated, which in turn transcriptionally regulates expression of p48 and XPC [38, 39]. In addition, the direct binding of p53 to the NER factors XP group B and D (XPB and XPD) has been demonstrated to inhibit their helicase activity, and may be involved in p53-mediated apoptosis and DNA repair pathways [40, 41]. As discussed in detail below, we have recently shown that overexpression of BRCA1 may compensate for loss of p53 in maintaining GGR through upregulation of XPC, DDB2, and GADD45 [42]. Other genes in addition to p53 may regulate expression of early DNA damage recognition factors in NER and help maintain NER when key regulators such as p53 are deficient in the cell.
BRCA1, p53, and NER To ascertain whether BRCA1 plays a role in NER we evaluated the effect of overexpression of BRCA1 protein on GGR and TCR in a human tumor cell line and determined whether this effect is p53 dependent. We demonstrated that overexpression of BRCA1 enhances GGR and induces p53-independent expression of p48, XPC, and GADD45 [42]. Specifically, we evaluated the effect of overexpression of BRCA1 protein on NER of UV-C irradiation induced photoproducts using human osteosar-
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coma tumor cells either wildtype or null for p53 allowing for regulated expression of BRCA1 [43]. Overexpression of BRCA1 significantly increased GGR but not TCR of UV photoproducts in cells null or wildtype for p53 [42]. To determine the mechanism for the effect of BRCA1 on GGR we analyzed the effect of induction of BRCA1 on expression and activity of NER genes involved in early steps of DNA photoproduct recognition and repair in genomic DNA. We found that induction of BRCA1 results in the increased expression of the XPC, DDB2, and GADD45 genes, the products of which are all involved in the initial DNA damage recognition processes, independently of p53. Both human and mouse studies have demonstrated that loss of XPC, DDB2, or GADD45 results in defective GGR, but normal TCR, consistent with the BRCA1 phenotype [36, 38, 39]. Our data and those of others suggest that BRCA1 affects DNA repair through transcriptional regulation of DNA damage recognition genes. Recent reports have suggested a mechanism for this regulation for GADD45; in the normal cellular state BRCA1 binds with the transcriptional coactivator CtIP and corepressor ZBRK1 and represses transcription of GADD45 [44]. After DNA damage BRCA1 and CtIP are phosphorylated by kinases, including ATM and ATR, and consequently dissociate and allow BRCA1 activation of GADD45 [45]. BRCA1 can activate the GADD45 promoter through its transactivation domain, and this effect is independent of p53, supporting an effect of BRCA1mediated activation of GADD45 on GGR in p53-deficient cells [46]. Undoubtedly the independent effects of both p53 and BRCA1 on the expression of p48, XPC, and GADD45 are complex and will require more sophisticated genetic models to completely understand their effects. Takimoto and colleagues [47] have suggested that BRCA1 affects NER in a p53-dependent manner. They found that BRCA1 can induce expression of DDB2 in cells that are wildtype for p53 and demonstrated that loss of BRCA1 expression delays repair of cyclobutane pyrimidine dimers using a host cell reactivation assay and a qualitative immunoflouresence assay. We also observed an effect on GGR with overexpression of BRCA1 in p53 wildtype cells, but this effect was less dramatic than what we observed in p53 deficient cells. In contrast to our findings, they did not observe DDB2 induction with overexpression of BRCA1 in p53-deficient cells. One explanation for this discrepancy is that Takimoto et al. used an adenovirus vector to overexpress BRCA1 in cells that contain mutant p53, whereas we used cells that expressed HPVE6 which targets p53 protein for degradation, essentially making these cells null for p53. Mutant p53 may interfere with an effect of BRCA1 on DDB2 transcription. Alternatively, BRCA1 may be acting as a coactivator of p53 and stabilizes or activates the small amount of residual p53 in the HPVE6 cells, resulting in induction of DDB2 and other DNA repair genes and maintenance of GGR. Both studies point to a role for BRCA1 in NER through transcriptional activation of
early DNA damage recognition genes. Further exploration will require identifying and using cell lines with endogenous BRCA1 and p53 mutations. Other investigators have suggested that the BRCA1 gene product is involved in TCR of oxidative DNA damage. Abbott and colleagues [48] demonstrated that mouse embryo fibroblasts null for BRCA1 and human cells with a homozygous deletion of BRCA1 are sensitive to IR and lack the ability to repair oxidative DNA damage caused by IR. However, a clinical correlation is lacking as BRCA1-associated breast cancers are not hypersensitive to IR [49]. Gowen et al. [50] suggested that murine embryonic stem cells null for the brca1 gene are deficient in TCR of IR induced oxidative DNA damage, but not UV-induced damage compared to parental cells. However, this paper was recently retracted on technical grounds, although the conclusions may still be valid [51]. Indeed other investigators have published data that demonstrate a role for BRCA1 in TCR of 8-oxoguanine lesions in human cells [52]. Therefore the role of BRCA1 in the repair of oxidative DNA damage remains unclear.
Functions of BRCA1 BRCA1 has been implicated in many other critical cellular processes in addition to NER, including DNA repair, cell cycle checkpoints, apoptosis, and proteosomal-mediated protein degradation [53, 54, 55, 56]. Several studies suggest that BRCA1 regulates these processes through protein-protein interactions. BRCA1 coimmunoprecipitates with a number of DNA repair proteins including MSH2, MSH6, ATM, RAD51, and the RAD50-MRE11NBS1 protein complex and localizes to nuclear foci with these proteins after treatment with DNA damaging agents, including IR and UV radiation [57, 58, 59]. In response to DNA damage BRCA1 is differentially phosphorylated at specific residues by phosphokinases including ATR after UV irradiation, and ATM and Chk2 after IR [45, 60]. BRCA1 encodes a 220-kDa nuclear phosphoprotein with several known structural motifs, including an N-terminal RING finger domain with an E3 ubiquitin ligase activity and two BRCT domains that bind the DNA strand-break repair complex Mre11/Rad50/Nbs1 [56, 61]. The BRCA1 RING domain forms a heterodimeric complex with the RING domain of BARD1 [54]. This complex confers a ubiquitin ligase function and undergoes auto-ubiquitination in vitro [54]. Other targets of the BRCA1/BARD1 ubiquitin ligase are currently under investigation and include Fanconi anemia (FA) complementation group D2 which undergoes mono-ubiquitination in a BRCA1-dependent manner after IR and UV [62, 63]. FA patients are predisposed to many kinds of cancer including squamous cell cancer of the head and neck, and FA cells are sensitive to crosslinking agents including mitomycin C and cisplatin. Cisplatin also induces intrastrand crosslinks, repaired by NER. Therefore BRCA1 and FA proteins may contribute
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to the repair of both intrastrand and interstrand crosslinks caused by exposure to chemotherapy agents including cisplatin and mitomycin C [64], suggesting possible therapeutic approaches to tumors deficient in this pathway. The association of BRCA1 with RAD51 suggests a role for BRCA1 in double strand break repair. Strong data exist implicating BRCA1 in homologous recombination; brca1 null mouse embryo fibroblasts have a defect in homologous recombination and a reduced level of gene targeting [65, 66] and human cells containing mutant BRCA1 have a decreased level of double strand break repair [67]. BRCA1 has been shown to localize to nuclear foci predominantly in S-phase with several other DNA repair proteins including MRE11/RAD50/NBS1 and BACH1 [61, 68]. BACH1 belongs to a family of DEAH helicases and has been found to bind to carboxyl BRCT repeats of BRCA1 and loss of function results in a decrease in double strand break repair [68]. The specificity and significance of many of these protein complexes and their role in DNA repair remains to be elucidated. One theory is that BRCA1 helps direct these proteins to double strand breaks. The data on a role for BRCA1 in nonhomologous end-joining is more controversial with some studies supporting a role and others not [69, 70, 71, 72, 73]. A role for BRCA1 in maintaining chromosomal stability fits with these data that BRCA1-deficient cells have a defect in error-free homologous recombination, but intact error prone nonhomologous DNA repair. Loss of NER would also contribute to a mutator phenotype in BRCA1-deficient cells. Over time loss of appropriate DNA repair will lead to mutagenesis of additional cancer genes and chromosomal instability in the cell and the subsequent development of malignancy. BRCA1 may regulate cellular processes through transcriptional coactivation. BRCA1 has a C-terminal transactivating domain [54, 59, 61, 74, 75] that has been found to stimulate transcription from the p21, p53, bax, and GADD45 promoters [46, 76]. BRCA1 has been found to complex with RNA polymerase II and has been shown to regulate transcription of GADD45 through interactions with the transcriptional coactivator CtIP and corepressor ZBRK1 [44, 59]. The data discussed here clearly point to a role for BRCA1 in many types of DNA repair, and loss of more than one of these functions of BRCA1 may be required for tumor suppression.
Clinical significance Similar to p53, but independently of it, BRCA1 contributes to the efficiency of GGR and regulates the expression of a set of NER genes. Strong clinical support for a role of BRCA1 in NER comes from mutation spectrum analysis of p53 in BRCA1 mutant tumors. Genetic evidence for a deficiency in the GGR pathway affected by BRCA1 has been described. Greenblatt et al. [28] report-
Fig. 3 Model for BRCA1-associated carcinogenesis. Loss of heterozygosity for BRCA1 leads to an initial decline in NER activity and potentially loss of ER, resulting in an accumulation of additional mutations, particularly arising from the nontranscribed strand of p53. Promoter hypermethylation of BRCA1 causes gene silencing and can also lead to a decline in GGR and mutations arising from the nontranscribed strand of p53. The breast epithelium may appear atypical after these changes have occurred. Loss of p53 function leads to even more pronounced genomic instability, resulting in additional genetic alterations leading to an invasive cancer phenotype
ed the type and location of p53 mutations in BRCA1/2 mutated breast tumors and compared the mutation spectrum to sporadic breast tumors. An increase in mutations arising from the nontranscribed strand of the p53 gene were identified in BRCA-associated tumors vs. sporadic tumors, and even more surprising was the identification of CCÆTT transversions. These mutations are seen almost exclusively in skin tumors harboring a defective NER pathway. These data suggest that p53 mutations in BRCA1-associated cancers occur after loss of heterozygosity of BRCA1. These data suggest a model for the involvement of BRCA1 and p53 in BRCA1-associated breast cancers (Fig. 3). Loss of heterozygosity for BRCA1 leads to an initial decline in NER activity and potentially loss of estrogen receptor (ER), resulting in an accumulation of additional mutations, specifically resulting in mutations of the nontranscribed strand of the p53 gene. Loss of p53 function leads to even more pronounced genomic instability, resulting in additional genetic alterations leading to an invasive cancer phenotype. This model presents a testable hypothesis for the multistep process leading to breast carcinogenesis, if relevant early premalignant lesions can be identified and sampled. These early premalignant lesions can be evaluated for the presence or absence of DNA repair genes and expression of breast specific markers such as the ER. Once these temporal events are defined, early targets of prevention can be developed. The translation of these results to the clinical setting is essential to define a role of NER in breast carcinogenesis.
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BRCA1 breast cancers and the estrogen receptor A current area of intense investigation is to understand why BRCA1 breast cancers are ER negative. One possible explanation is that loss of BRCA1 somehow leads to downregulation of the ER at a specific time in the pathway of the development of these cancers. Data supporting ligand independent activation of the ER in BRCA1 deficient cells exists, but the link to loss of ER expression has not been established [77]. Another plausible explanation is that loss of BRCA1 allows expression of genes that would otherwise be silenced through its role in heterochromatinizing XIST RNA [78]. It will be important to try and determine when, if ever, ER is present on the breast epithelium in these individuals. If the ER is present on the breast epithelium but at an earlier age than we would normally prescribe tamoxifen for risk reduction, these patients may benefit from earlier intervention. Presently it is unclear whether women who carry a BRCA1 mutation would benefit from tamoxifen, particularly given the fact that BRCA1-associated breast cancers are usually ER negative. However, women who carry a BRCA1 mutation have a 50% reduction in the risk of breast cancer if they have their ovaries removed premenopausally, suggesting that their breast epithelium is hormonally responsive in the premenopausal state. Perhaps giving estrogen blockade at a younger age would have preventative effects in BRCA1 carriers.
Chemosensitivity and clinical risk assessment The detection of NER gene expression levels in premalignant and cancerous breast lesions may be useful in risk assessment and may be able to help determine cancer chemosensitivity. BRCA1-mutant breast and ovarian cancer may be particularly sensitive to cisplatin due to defective DNA repair of both intrastrand and interstrand crosslinks. Indeed, BRCA1 null cells are hypersensitive to cisplatin in tissue culture [79, 80, 81]. The NER DNA damage recognition genes XPC and p48 play a major role in repair of intrastrand crosslinks. Whether these genes play a role in repair of interstrand crosslinks remains to be tested and may provide a possible link between BRCA1, NER, and FA. Levels of these damage recognition genes may allow us to predict which patients would benefit from cisplatin-based chemotherapy. If used in treatment of these cancers, cisplatin-based chemotherapy may confer a longer disease-free survival and overall survival than standard adjuvant chemotherapy. These investigations and ongoing studies into the role of NER and chemosensitivity in both mouse and human cells deficient for BRCA1 and the interaction of both p53 and BRCA1 in ATR-regulated UV-DNA damage pathways are underway in our laboratory. Expression levels, promoter methylation status, and polymorphisms of NER DNA damage recognition genes and FA complementation groups in screen detected breast lesions may allow us to predict who is going to
develop breast cancer and whom we should be screening more closely. We are currently assessing the role of these genes in the progression of normal breast epithelium to cancer in women who are undergoing screening and carry a BRCA1 mutation in our clinical breast cancer genetics program.
Conclusions Despite recent advances in understanding the cellular localization and many protein interactions of the BRCA1 gene product, the specific functional role of BRCA1 in normal breast and ovarian tissue remains unclear, as does the effect of its loss on tumorigenesis in these organs. We have identified a potential mechanism for the role of BRCA1 in NER through transcriptional regulation of DNA damage recognition genes. Identifying the exact role of BRCA1 in DNA repair pathways, its interaction with p53 in these pathways, and its effect on expression of DNA damage genes will shed light on the tumor suppressor functions of BRCA1 and suggest approaches to risk assessment and chemotherapeutic management of breast cancer. Acknowledgements A.R.H. was supported by an ASCO Young Investigator Award and a Postdoctoral Fellowship from the California Breast Cancer Research Program. J.M.F. was supported by the National Institutes of Health Award RO1 CA83889, a California Breast Cancer Research Program Pilot Award, a California Cancer Research Program Research Award, a V Foundation Award in Translational Science, and a Burroughs Wellcome Fund New Investigator Award in Toxicological Sciences.
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