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, 19 (7), 4633-42

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

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

N J Marston et al. Mol Cell Biol.

Abstract

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.

Figures

FIG. 1
FIG. 1
Diagrammatic representation of the human BRCA2 protein showing the regions used in this study. The section of the protein encoded by exon 11 is shaded, and the BRC repeats (6) are represented by filled arrows. The location of the N-terminal region reported to have transcriptional activity (30) and the Rad51-binding sites are indicated. The positions of the protein fragments expressed by the various constructs are shown beneath the diagram, with the extent indicated in amino acids.
FIG. 2
FIG. 2
Demonstration of an interaction between BRCA2 and DSS1 in yeast and mammalian two-hybrid systems. (A) Yeast two-hybrid interaction between human BRCA2.IV and DSS1, determined by β-Galactosidase filter assay of Y190 strains transformed with combinations of two-hybrid vectors encoding GAL4 DNA-binding domain (DBD) fusion partner and GAL4 activation domain (AD) fusion partner (human BRCA2 aa 2472 to 2957 [IV], BRCA2 aa 3215 to 3418 [XC], full-length human DSS1 cDNA clone [DSS1], full-length RAD51 cDNA clone [RAD51], or empty vector [−]). (B) Two-hybrid assay in human 293T cells. The indicated combinations of GAL4 DNA-binding domain (DBD) and VP16 activation domain (AD) fusions with BRCA2.IV and full-length human DSS1 or empty vector were transiently expressed in 293T cells with a GAL4-luciferase reporter, and activation was measured by luciferase assay on cell lysates. Data are presented as mean fold activation of the reporter above background from four separate experiments.
FIG. 3
FIG. 3
Coimmunoprecipitation of BRCA2 and DSS1 in mammalian cells. (A) DSS1 antisera can be used for specific immunoprecipitation. Preparations of GST or GST-DSS1 were immunoprecipitated in parallel with 1 or 10 μl of polyclonal antisera generated in this study. Antisera 286 and 287 were raised against the full-length recombinant DSS1, and antisera 290 and 291 were raised against a C-terminal peptide of DSS1. Immunoprecipitates were analyzed by immunoblotting with anti-GST antibody. The input proteins are shown. Sizes in both panels are indicated in kilodaltons. (B) Interaction of DSS1 with the C terminus of BRCA2 in transiently transfected COS cells. Lysates from COS cells transiently transfected with 9E10-tagged DSS1 (+) and various FLAG-tagged BRCA2 fragments (indicated by amino acid numbers) were immunoprecipitated with anti-Myc tag antibody 9E10, anti-DSS1 antiserum 290, or control anti-HA tag antibody 12CA5. Immunoprecipitates were analyzed by SDS-PAGE (6% polyacrylamide gel), together with samples of the input lysates representing half the protein used in the immunoprecipitations, followed by Western blot analysis with anti-FLAG tag antibody M2.
FIG. 4
FIG. 4
Coimmunoprecipitation of endogenous BRCA2 with DSS1 in MCF7 cells. Lysates of MCF7 cells cultured below confluency in 10% fetal calf serum, and lysates of COS cells transiently transfected with (+) or without (−) a construct expressing full-length (F.L.) BRCA2 were immunoprecipitated, in parallel, with anti-BRCA2 antiserum B, anti-DSS1 antiserum 290, or their preimmune sera (B PI and 290 PI). Immunoprecipitates were analyzed by SDS-PAGE (6% polyacrylamide gel) and Western blotting with an anti-BRCA2 rat monoclonal antibody.
FIG. 5
FIG. 5
Expression of DSS1 is serum stimulated. Immunofluorescence detection of DSS1 in MCF7 cells which had been starved of serum for 24 h (A) or subsequently refed with 10% fetal calf serum for 6 h (B) or 24 h (C and D). The staining pattern of the antiserum is specific, as shown by preblocking the antibody with the immunizing peptide (D), as opposed to a nonspecific peptide (A to C). Visualization was with fluorescein isothiocyanate conjugated goat anti-rabbit immunoglobulin G.
FIG. 6
FIG. 6
Yeast orthologues of DSS1. Alignment of human/mouse DSS1 protein sequence (hu/mo) (13) with amino acid sequences of putative products of DSS1-like open reading frames in S. pombe (sp) and S. cerevisiae (sc). Identical and conserved residues are indicated by solid and broken bars, respectively.
FIG. 7
FIG. 7
Construction of deletions of yeast DSS1 genes. (A) S. cerevisiae DSS1 deletion. The 5′ and 3′ genomic fragments flanking the DSS1 gene which were used in the deletion construct, shown in the wild-type and Δdss1 arrangements, with the extent of the deletion in nucleotides indicated beneath in kilobases. Haploid clones derived by sporulation of cells transformed with the deletion cassette were analyzed for DSS1 status by PCR on genomic DNA. Positions of forward and reverse primers 1 and 2 in the genomic sequences flanking those used in the deletion cassette are indicated in the upper panel with the expected size of the PCR product for wild-type or deleted clones. PCR results for three tetrads (3.1, 3.2, and 3.3) and the parent strain are shown at the bottom. (B) S. pombe dss1 deletion cassette, illustrating the 5′ and 3′ genomic fragments used to construct the deletion cassette, in both wild-type and Δdss1 configurations. Confirmation of the deletion by PCR is shown at the bottom.
FIG. 8
FIG. 8
Yeast Δdss1 phenotypes. (A) Temperature-sensitive growth of S. pombe and S. cerevisiae deleted for DSS1. Equivalent cultures of wild-type and Δdss1 yeast clones were diluted to the same concentration and serially diluted 1:10. These dilutions of each yeast were plated in series, and the plates were incubated at 25, 30, or 35°C, as indicated. (B) Morphological phenotypes of cells from exponentially growing cultures of the following S. pombe strains: 1, wild type; 2, Δdss1; 3, Δdss1 expressing human DSS1; 4, Δdss1 expressing S. pombe dss1+. (C) Rescue of the temperature-sensitive growth of Δdss1 S. pombe at 35°C by expression of human DSS1 (hDSS1) or S. pombe dss1+ (spdss1).
FIG. 9
FIG. 9
DNA content of Δdss1 S. pombe. (A) The cell cycle distributions of wild-type and Δdss1 S. pombe strains were examined by flow cytometry using PI staining of fixed cells from cultures grown at 30 and 35°C for 6 h. The data for 2 × 104 ungated events are presented as relative DNA content on the horizontal axis and cell number on the vertical axis. (B) Microscopic images of PI-stained cells of wild-type (image 1) and Δdss1 (image 2) strains of S. pombe, prepared as for FACS analysis.

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