Absence of Brca2 causes genome instability by chromosome breakage and loss associated with centrosome amplification

AbstractWomen heterozygous for mutations in the breast-cancer susceptibility genes BRCA1 and BRCA2 have a highly elevated risk of developing breast cancer [1]. BRCA1 and BRCA2 encode large proteins with no sequence similarity to one another. Although involvement in DNA repair and transcription has been suggested, it is still not understood how loss of function of these genes leads to breast cancer [2]. Embryonic fibroblasts (MEFs) derived from mice homozygous for a hypomorphic mutation (Brca2Tr2014) within the 3′ region of exon 11 in Brca2[3], or a similar mutation (Brca2Tr) [4], proliferate poorly in culture and overexpress the tumour suppressor p53 and the cyclin-dependent kinase inhibitor p21Waf1/Cip1. These MEFs have intact p53-dependent DNA damage G1–S [3,4] and G2–M checkpoints [4], but are impaired in DNA double-strand break repair [3] and develop chromosome aberrations [4]. Here, we report that Brca2Tr2014/Tr2014 MEFs frequently develop micronuclei. These abnormal DNA-containing bodies were formed through both loss of acentric chromosome fragments and by chromosome missegregation, which resulted in aneuploidy. Absence of Brca2 also led to centrosome amplification, which we found associated with the formation of micronuclei. These data suggest a potential mechanism whereby loss of BRCA2 may, within subclones, drive the loss of cell-cycle regulation genes, enabling proliferation and tumourigenesis

Interaction between the Product of the Breast Cancer Susceptibility Gene BRCA2 and DSS1, a Protein Functionally Conserved from Yeast to Mammals

Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle

N-terminal polyubiquitination and degradation of the Arf tumor suppressor

Unknown mechanisms govern degradation of the p19(Arf) tumor suppressor, an activator of p53 and inhibitor of ribosomal RNA processing. Kinetic metabolic labeling of cells with [(3)H]-leucine indicated that p19(Arf) is a relatively stable protein (half-life ∼6 h) whose degradation depends upon the ubiquitin–proteasome pathway. Although p19(Arf) binds to the Mdm2 E3 ubiquitin protein ligase to activate p53, neither of these molecules regulates p19(Arf) turnover. In contrast, the nucleolar protein nucleophosmin/B23, which binds to p19(Arf) with high stoichiometry, retards its turnover, and Arf mutants that do not efficiently associate with nucleophosmin/B23 are unstable and functionally impaired. Mouse p19(Arf), although highly basic (22% arginine content), contains only a single lysine residue absent from human p14(ARF), and substitution of arginine for lysine in mouse p19(Arf) had no effect on its rate of degradation. Mouse p19(Arf) (either wild-type or lacking lysine) and human p14(ARF) undergo N-terminal polyubiquitination, a process that has not as yet been documented in naturally occurring lysine-less proteins. Re-engineering of the p19(Arf) N terminus to provide consensus sequences for N-acetylation limited Arf ubiquitination and decelerated its turnover

Cell cycle and genetic background dependence of the effect of loss of BRCA2 on ionizing radiation sensitivity

Cell cycle and genetic background dependence of the effect of loss of BRCA2 on ionizing radiation sensitivity. Carriers of mutations in the BRCA2 gene are at a highly elevated risk of breast and other cancers. The BRCA2 gene encodes a very large protein thought to play a role in DNA repair. To examine the effect of mutation of BRCA2 on sensitivity to ionizing radiation, we used a previously described mouse model system (Brca2(Tr)) in which the Brca2 open reading frame is truncated. Mouse embryo fibroblasts carrying this mutation have a proliferative defect, which we show here can be substantially rescued by genetic ablation of p53. Proliferating Brca2(Tr/Tr)/p53(-/-) cells, like Brca2(Tr/Tr) cells, show genomic instability. We used the clonogenic survival assay, which depends on the ability of cells to proliferate, to examine the cell cycle dependence of radiation sensitivity of Brca2(Tr/Tr)/p53(-/-) compared to p53(-/-) and wild-type cells. This showed that the Brca2 mutation had little effect on cells irradiated in quiescence but sensitized proliferating cells to ionizing radiation on a p53(-/-) background. These results suggest that the major role of Brca2 in mediating cell survival after irradiation is in the S and G(2) phases of the cell cycle