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

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.

DSS1 is required for the stability of BRCA2

“…Characterization of the endogenous DSS1 protein Previous studies have shown that endogenous human and mouse DSS1 cannot be detected by Western blotting, although both recombinant and yeast DSS1 are detectable by this method (Jantti et al, 1999;Marston et al, 1999;Wei et al, 2003;Gudmundsdottir et al, 2004;Isono et al, 2004;Sone et al, 2004). To detect endogenous human DSS1, we generated an anti-DSS1 antiserum against a full-length DSS1 GST-fusion protein.…”

“…Deletion of DSS1 orthologues in Schizosaccharomyces pombe and Saccharomyces cerevisiae leads to reduced growth rate especially at low and high temperatures. Notably, Ddss1 cells exhibit polyploid DNA content at elevated temperatures (Marston et al, 1999).…”

“…It has been reported that DSS1 (deleted in spilt hand/ spilt foot) binds to BRCA2 in the region of residues 2472-2957, which is the longest conserved region in the BRCA2 protein (Marston et al, 1999). However, the mechanism by which DSS1, an evolutionarily conserved protein of unknown function, participates in BRCA2 function remains unclear.…”

“…This has not been possible before, a fact that has been an obstacle in previous studies (Marston et al, 1999;Yang et al, 2002;Gudmundsdottir et al, 2004). We then used the RNAi strategy to examine the consequence of DSS1 depletion.…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…The BRCA2 C-terminus forms a tight complex with a conserved protein, DSS1, and can also interact with Rad51 [19,71,137]. The crystal structure of the BRCA2 C-terminus bound to DSS1 revealed similarity with the heterotrimeric ssDNA-binding complex, RPA, implying a direct interaction of BRCA2/DSS1with ssDNA [138].…”