A Naturally Occurring Allele of BRCA1 Coding for a Temperature-Sensitive Mutant Protein

Worley, Terri; Vallon‐Christersson, Johan; Billack, Blasé; Borg, Åke; Monteiro, Alvaro N.A.

doi:10.4161/cbt.1.5.164

Cited by 24 publications

(21 citation statements)

References 20 publications

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“…More than 70 missense substitutions have been recorded that alter the primary sequence of the tandem BRCT repeats, but pedigree analysis clarifying the disease linkage of these alleles is available for only eight of these variants (6,7,12,(23)(24)(25)(26)(27). Many of these amino acid substitutions may be linked with disease but remain as unclassified in the BIC, because the presence of the allele has not been tested in the general population, or the segregation of the allele with disease within a family is unclear (6, 7).…”

mentioning

confidence: 99%

Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations

Williams

Chasman

Hau

et al. 2003

Journal of Biological Chemistry

123

141

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Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein, highlighting a crucial role for the C-terminal, BRCT repeat region in mediating BRCA1 tumor suppressor function. However, the molecular and genetic effects of missense mutations that map to the BRCT region remain largely unknown. Using a protease-based assay, we directly assessed the sensitivity of the folding of the BRCT domain to an extensive set of truncation and single amino acid substitutions derived from breast cancer screening programs. The protein can tolerate truncations of up to 8 amino acids, but further deletion results in drastic BRCT folding defects. This molecular phenotype can be correlated with an increased susceptibility to disease. A cross-validated computational assessment of the BRCT mutation data base suggests that as much as half of all BRCT missense mutations contribute to BRCA1 loss of function and disease through protein-destabilizing effects. The coupled use of proteolytic methods and computational predictive methods to detect mutant BRCA1 conformations at the protein level will augment the efficacy of current BRCA1 screening protocols, especially in the absence of clinical data that can be used to discriminate deleterious BRCT missense mutations from benign polymorphisms.Germline mutations within the breast and ovarian cancer susceptibility gene BRCA1 predispose carriers to early-onset breast and breast-ovarian cancers (1). Accumulating evidence points to a role for the BRCA1 protein product in the regulation of multiple nuclear functions including transcription, recombination, DNA repair, and checkpoint control (2-4). Tumor-associated mutations occur throughout the BRCA1 coding sequence, but cluster to sequences encoding the N-terminal RING finger domain and the two carboxy-terminal repeat BRCT 1 domains (5-7).The molecular details of how BRCA1 mutations contribute to the pathogenesis of cancer remain largely unknown. The functional significance of the BRCT region is highlighted by the high degree of sequence conservation within the BRCT regions of among mammalian, Xenopus, and avian BRCA1 homologues (8 -10). Several lines of evidence reveal the BRCT is required for tumor suppressor function. A nonsense mutation, which removes 11 C-terminal residues of the second, BRCT (Tyr 1853 3 stop), is associated with early-onset breast cancer (11). Two cancer-linked BRCT missense mutations (12) that destabilize the BRCT fold (13-15), A1708E and M1775R, ablate the double-strand break repair and transcription function of BRCA1 (16) and inhibit BRCT interactions with histone deacetylases (17), BACH1 (18), and the transcriptional co-repressor CtIP (19,20). Furthermore, mice with homozygous targeted mutations removing the C-terminal half of BRCA1 are viable but develop tumors, suggesting the missing BRCT and/or other domains are expendable for survival, but not for tumor suppression (21).Although all frameshift or nonsense mutations recorded in the Breast cancer Information Cor...

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mentioning

confidence: 99%

Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations

Williams

Chasman

Hau

et al. 2003

Journal of Biological Chemistry

123

141

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“…The aims in this proposal were performed by two fellows: Blase Billack (2002 and who left for a faculty position in breast cancer research and Vesna Dapic (2004). During our characterization of the R1699W mutant and of the other temperature-sensitive mutants isolated in yeast 25, 26 we found that these mutants were not yet widely applicable as tools.…”

Section: Bodymentioning

confidence: 99%

Characterization and Use of Temperature-Sensitive Mutations of BRCA1 for the Study of BRCA1 Function

Billack¹,

Monteiro²,

Dapíc³

2005

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Public reporting burden for this collection of information is estimated to average I hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and ABSTRACT (Maximum 200 Words)Recent evidence suggests that the breast and ovarian cancer susceptibility gene product BRCA1 is involved in the DNA damage response. However, the precise biochemical function of BRCA1 has remained unknown. Initially, we identified a naturally-occurring allele of BRCA1 which codes for a single amino acid substitution from arginine to tryptophan at residue 1699 (R1699W) and displays temperaturesensitive activity in transcription. The transcription activity of the mutant protein appears to undergo complex regulation, as temperature-sensitive (TS) transcription activation was found to be cell type specific and not dependent on the tissue of origin. We have characterized the behavior of this mutant and isolated several stable cell lines. In addition, through a screen in yeast we identified 11 more unique TS mutants and determined a preferential location for these mutations in the first BRCT domain of BRCA1.We have also generated a series of mutations at residue 1699 and characterized them for transcription activity in different temperatures. Our results provide a framework to engineer conditional mutants of BRCA1 that will aid in the dissection of its in vivo function. SUBJECT TERMS NUMBER OF PAGESCancer biology, BRCA1, transcription activation, gene expression, 52 INTRODUCTIONLinkage analysis revealed that mutations in the BRCA1 locus account for the majority of families with multiple members affected with breast and ovarian cancer 1. The cloning and sequencing of BRCA1 did not immediately yield clues to its cellular function(s). The gene codes for a product with little resemblance to other 2 proteins of known function . The observation that BRCA1 colocalized to Rad5l foci during S phase and that these foci underwent a dynamic reorganization following DNA damaging insults provided the first link to the DNA damage response pathway . It soon became evident that BRCA1 played an important role in the cellular response to DNA damage because BRCA1 deficiency compromised homologous recombination, transcriptioncoupled repair, microhomology end-joining, and non-homologous end-joining 5-10. Taken together, these data firmly placed BRCA1 in the DNA damage response pathway 1 1 but its precise biochemical role has continued to elude researchers.In the 10 years since it was cloned, BRCA1 has been proposed to play a role in ubiquitination 12,13 chromatin remodeling [14][15][16] and several aspects of transcriptio...

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“…The pathogenicity of only a small number of these variants has been inferred genetically [12][13][14][15][16] or tested functionally [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. Underscoring the interest in and need for accurate classification of sequence variants in BRCA1 are recent descriptions of novel approaches to predict the pathogenicity of non-synonymous amino acid substitutions.…”

Section: Introductionmentioning

confidence: 99%

“…A multiple sequence alignment of fulllength BRCA2 cDNA sequences was generated using the T-coffee program (29). The alignment was restricted to BRCA2 cDNA sequences derived from vertebrates in an effort to avoid using sequences that are evolutionarily 4 S.V.…”

Section: Introductionmentioning

confidence: 99%

“…There is a growing interest in developing efficient and reliable ways to classify the pathogenicity of these variants and a variety of approaches have been reported recently. These include analyses of patterns of cosegregation [9], assessment of variant frequency in unaffected controls [6], predictions based on the position and nature of the amino acid change [7] and biochemical and functional assays [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of BRCA2 Mutation in a Series of Functional Assays

Couch¹

2005

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Public reporting burden for this collection of information is estimated to average I hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE (DD-MM-YYYY)2. REPORT TYPE 3. DATES COVERED (From -To) 5f. WORK UNIT NUMBER PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERMayo Clinic Rochester Rochester, MN 55905 SPONSORING I MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S)U BodyAim #1: To assess the role of BRCA2 in cell growth.We proposed to establish Capan-1 BRCA2 mutant cells that stably express wildtype BRCA2. However, using multiple different transfection techniques we were unable to achieve greater than 0.01% transfection efficiency, which is insufficient for the outlined experiments. To overcome this problem we proposed to overexpress BRCA2 in MCF7 breast cancer cells because the cells were readily transfected and because overexpression of mutant forms could competitively inhibit wildtype BRCA2 function. We focused our efforts on the use of MCF7 cells stably expressing wtBRCA2, vector alone, and 6174deIT-BRCA2. Expression was confirmed by western blot (Task 1). We also tried to generate MCF7 Tet-On and MCF7 Tet-Off inducible BRCA2 cell lines to no avail (Task 2 and 6). The stably expressing MCF7 cells were subsequently assessed for alterations in growth rate (MTT assays), anchorage independence (soft agar assays), colony formation, and cell cycle (FACS) (Task 3). In MTT assays two independent wtBRCA2 expressing cell lines grew significantly slower than vector transfected cells, while cells expressing mutant BRCA2 grew the most rapidly. In soft agar and colony formation assays more colonies formed from mutant or vector expressing cells. Cell cycle analysis revealed no change in the cell cycle over time. Overall, the data suggested that BRCA2 regulates cell growth and transformation in a cell cycle independent manner.A total of 8 BRCA2 mutant constructs containing 7 missense mutations and 1 truncating mutation (K3326X) in different domains of BRCA2 were generated. The Y42C mutation is located in a putative transactivation domain, while E462G, P655R, 4812C>T, and K1690N are all located in the BRC repeat region that has been associated with DNA repair activity, and the D2723...

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A Naturally Occurring Allele of BRCA1 Coding for a Temperature-Sensitive Mutant Protein

Cited by 24 publications

References 20 publications

Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations

Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations

Characterization and Use of Temperature-Sensitive Mutations of BRCA1 for the Study of BRCA1 Function

Characterization of BRCA2 Mutation in a Series of Functional Assays

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