Functional Characterization of BRCA1 Sequence Variants using a Yeast Small Colony Phenotype Assay

Germline mutations that inactivate the tumor suppressor gene BRCA1 are associated with an increased risk of cancers of the breast and other tissues, but the functional consequence of many missense variants found in the human population is uncertain. Several predictive methods have been proposed to distinguish cancer-predisposing missense mutations from harmless polymorphisms, including a small colony phenotype (SCP) assay performed in the model organism, yeast (Saccharomyces cerevisiae). The goal of this study was to further evaluate this colony size assay. We constructed 28 missense mutations throughout the C-terminal 305 amino acid residues of BRCA1. Mutated proteins were expressed in yeast and evaluated using the SCP assay. We conclude there is as yet no evidence the assay can identify inactivating mutations upstream of the BRCT repeats. However, within and between the BRCT repeats, results of the assay are in general agreement with predictions based on structural modeling, other in vitro and in vivo assays, and cross-species sequence conservation. Thus, the yeast assay appears to provide confirmatory in vivo evidence to aid in characterizing some BRCA1 missense variants.

[1]  Thomas Ludwig,et al.  The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity. , 2002, Current opinion in genetics & development.

[2]  J. Manley,et al.  Functional interaction of BRCA1-associated BARD1 with polyadenylation factor CstF-50. , 1999, Science.

[3]  G M Lenoir,et al.  Hereditary breast cancer: Pathobiology, prognosis, and BRCA1 and BRCA2 gene linkage , 1996, Cancer.

[4]  A. Monteiro,et al.  BRCA1: exploring the links to transcription. , 2000, Trends in biochemical sciences.

[5]  A. Monteiro,et al.  Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation. , 2000, Cancer research.

[6]  D. Easton,et al.  Breast and ovarian cancer incidence in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. , 1995, American journal of human genetics.

[7]  F. Couch,et al.  BRCA1 Facilitates Stress-induced Apoptosis in Breast and Ovarian Cancer Cell Lines* , 2000, The Journal of Biological Chemistry.

[8]  F. Collins,et al.  Human BRCA1 inhibits growth in yeast: potential use in diagnostic testing. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[9]  I. Verma,et al.  Transcriptional activation by BRCA1 , 1996, Nature.

[10]  M. Sternberg,et al.  Structure of an XRCC1 BRCT domain: a new protein–protein interaction module , 1998, The EMBO journal.

[11]  R. Jensen,et al.  BRCA1 Expression Restores Radiation Resistance in BRCA1-defective Cancer Cells through Enhancement of Transcription-coupled DNA Repair* , 1999, The Journal of Biological Chemistry.

[12]  A. Monteiro,et al.  Yeast-based assays for detection and characterization of mutations in BRCA1. , 1998, Breast disease.

[13]  M. Sternberg,et al.  The BRCA1 C-terminal domain: structure and function. , 2000, Mutation research.

[14]  A. Vojtek,et al.  Ras-Raf interaction: two-hybrid analysis. , 1995, Methods in enzymology.

[15]  R. Carter,et al.  BRCA1, BRCA2 and breast cancer: a concise clinical review. , 2001, Clinical and investigative medicine. Medecine clinique et experimentale.

[16]  D. Livingston,et al.  Structure of the 53BP1 BRCT region bound to p53 and its comparison to the Brca1 BRCT structure. , 2002, Genes & development.

[17]  S. Neuhausen,et al.  Loss of heterozygosity in familial tumors from three BRCA1-linked kindreds. , 1994, Cancer Research.

[18]  Yanfen Hu,et al.  Chromatin remodeling and activation of chromosomal DNA replication by an acidic transcriptional activation domain from BRCA1. , 1999, Genes & development.

[19]  D. Easton,et al.  Genetic linkage analysis in familial breast and ovarian cancer: results from 214 families. The Breast Cancer Linkage Consortium. , 1993, American journal of human genetics.

[20]  A. Ashworth,et al.  New complexities for BRCA1 and BRCA2 , 2001, Current Biology.

[21]  Steven E. Bayer,et al.  A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. , 1994, Science.

[22]  R. White,et al.  BRCA1 is associated with the centrosome during mitosis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Glover,et al.  Structural Consequences of a Cancer-causing BRCA1-BRCT Missense Mutation* , 2003, The Journal of Biological Chemistry.

[24]  Yudong D. He,et al.  Gene expression profiling predicts clinical outcome of breast cancer , 2002, Nature.

[25]  J. Parvin,et al.  Factors associated with the mammalian RNA polymerase II holoenzyme. , 1998, Nucleic acids research.

[26]  I. Hedenfalk,et al.  Gene expression profiling of hereditary and sporadic ovarian cancers reveals unique BRCA1 and BRCA2 signatures. , 2002, Journal of the National Cancer Institute.

[27]  E. Liu,et al.  BRCA1-associated growth arrest is RB-dependent. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Y. Houvras,et al.  Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiPl , 1997, Nature.

[29]  P. Johnston,et al.  BRCA1: mechanisms of inactivation and implications for management of patients , 2002, The Lancet.

[30]  W. El-Deiry,et al.  BRCA1 Effects on the Cell Cycle and the DNA Damage Response Are Linked to Altered Gene Expression* , 2000, The Journal of Biological Chemistry.

[31]  P. Møller,et al.  Functional analysis of BRCA1 C-terminal missense mutations identified in breast and ovarian cancer families. , 2001, Human molecular genetics.

[32]  D. Livingston,et al.  Functional communication between endogenous BRCA1 and its partner, BARD1, during Xenopus laevis development , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Potter,et al.  Understanding missense mutations in the BRCA1 gene: An evolutionary approach , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Nebojsa Mirkovic,et al.  Structure-Based Assessment of Missense Mutations in Human BRCA1 , 2004, Cancer Research.

[35]  L. Itzhaki,et al.  Characterisation of the BRCT domains of the breast cancer susceptibility gene product BRCA1. , 2002, Journal of molecular biology.

[36]  F. Couch,et al.  Mutations and Polymorphisms in the familial early‐onset breast cancer (BRCA1) gene , 1996, Human mutation.

[37]  D. Livingston,et al.  Genetic analysis of BRCA1 function in a defined tumor cell line. , 1999, Molecular cell.

[38]  T. Miyake,et al.  A Functional Comparison of BRCA1 C-terminal Domains in Transcription Activation and Chromatin Remodeling* , 2000, The Journal of Biological Chemistry.

[39]  C. Deng,et al.  A targeted disruption of the murine Brca1 gene causes γ-irradiation hypersensitivity and genetic instability , 1998, Oncogene.

[40]  M. Stratton.  Pathology of familial breast cancer: differences between breast cancers in carriers of BRCA1 or BRCA2 mutations and sporadic cases , 1997, The Lancet.

[41]  J. Glover,et al.  Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1 , 2001, Nature Structural Biology.

[42]  A. Zharkikh,et al.  Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation , 2004, Journal of Medical Genetics.

[43]  D. Chasman,et al.  Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations* , 2003, Journal of Biological Chemistry.

[44]  H. Hanafusa,et al.  Evidence for a transcriptional activation function of BRCA1 C-terminal region. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[45]  R. Scully,et al.  BRCA1 and BRCA2 in hereditary breast cancer. , 2002, Biochimie.

[46]  E. Rosen,et al.  BRCA1 and Prostate Cancer , 2001, Cancer investigation.

[47]  B. Koller,et al.  Brca1 controls homology-directed DNA repair. , 1999, Molecular cell.

[48]  M. King,et al.  Human, canine and murine BRCA1 genes: sequence comparison among species. , 1996, Human molecular genetics.

[49]  M. Stratton,et al.  Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations. , 1998, Journal of the National Cancer Institute.

[50]  X. Wang,et al.  Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. , 1999, Molecular cell.

[51]  B. Ponder,et al.  Genetic testing for cancer risk. , 1997, Science.

[52]  L. Bennett,et al.  Sequence analysis of the rat Brca1 homolog and its promoter region , 1999, Mammalian Genome.

[53]  Patrick Dowd,et al.  Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families , 1994, Nature Genetics.

[54]  Alexander Kinev,et al.  BRCA1 Is Associated with a Human SWI/SNF-Related Complex Linking Chromatin Remodeling to Breast Cancer , 2000, Cell.

[55]  A. Venkitaraman,et al.  Functions of BRCA1 and BRCA2 in the biological response to DNA damage. , 2001, Journal of cell science.

[56]  H. Hanafusa,et al.  Common BRCA1 variants and transcriptional activation. , 1997, American journal of human genetics.

[57]  Anne E Carpenter,et al.  BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations , 2001, The Journal of cell biology.

[58]  Rachel E. Klevit,et al.  Structure of a BRCA1–BARD1 heterodimeric RING–RING complex , 2001, Nature Structural Biology.

[59]  B. Orelli,et al.  Nine novel conserved motifs in BRCA1 identified by the chicken orthologue , 2001, Oncogene.

[60]  D. Easton,et al.  Risks of cancer in BRCA1-mutation carriers , 1994, The Lancet.

[61]  R. Young,et al.  BRCA1 is a component of the RNA polymerase II holoenzyme. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[62]  D. Easton,et al.  Allele losses in the region 17q12–21 in familial breast and ovarian cancer involve the wild–type chromosome , 1992, Nature Genetics.