DSS1 is required for RAD51 focus formation and genomic stability in mammalian cells - PubMed (original) (raw)
DSS1 is required for RAD51 focus formation and genomic stability in mammalian cells
Katrin Gudmundsdottir et al. EMBO Rep. 2004 Oct.
Abstract
BRCA2 is a breast cancer susceptibility gene implicated in the repair of double-strand breaks by homologous recombination with RAD51. BRCA2 associates with a 70-amino-acid protein, DSS1, but the functional significance of this interaction has remained unclear. Recently, deficiency of a DSS1 orthologue in the fungus Ustilago maydis has been shown to cause a defect in recombinational DNA repair. Here we have investigated the consequences of DSS1 depletion in mammalian cells. We show that like BRCA2, DSS1 is required for DNA damage-induced RAD51 focus formation and for the maintenance of genomic stability, indicating a function conserved from lower eukaryotes to humans. However, DSS1 seems to be not required for BRCA2 or RAD51 stability or for BRCA2 and RAD51 to interact, raising the possibility that DSS1 may be required for the BRCA2-RAD51 complex to become associated with sites of DNA damage.
Figures
Figure 1
Silencing of DSS1 using RNAi leads to a defect in clonogenic capacity in mammalian cells. (A) Reduction of DSS1 expression by RNAi. Mouse embryonic stem (ES) cells and MCF7 cells were co-transfected with vectors expressing mouse or human DSS1–eGFP fusion proteins and vectors expressing eCFP and siRNAs specific for mouse or human DSS1 (pSUPER–eCFP–mDss1 and pSUPER–eCFP–hDSS1). Cells were also transfected with a control vector (pSUPER–eCFP–NS). At 24 h after transfection, cell lysates were prepared and analysed by western blotting. Blots were probed with anti-GFP antiserum, which recognizes both GFP and CFP. The CFP expressed from the pSUPER vector acts as a transfection and loading control. (B) Reduction of endogenous Dss1 mRNA levels measured by RT–PCR. ES cells transfected with pSUPER–mDss1 were analysed after 24 h and compared with Dss1 mRNA levels in ES cells transfected with pSUPER–NS and in untransfected cells. Levels of Gapdh were used as a control. (C) Colony formation was analysed in ES cells and MCF7 cells transfected with either pSUPER–NS or pSUPER–mDss1/pSUPER–hDSS1, respectively, together with a blasticidin resistance-encoding plasmid (pEF-Bsd). Transfection efficiencies were approximately 70%. Cells were maintained under antibiotic selection for 10–14 days and then stained with crystal violet and colonies counted. The graph shows the average percentage of colonies formed by cells transfected with pSUPER–mDss1/pSUPER–hDSS1 compared with pSUPER-NS-transfected cells (corrected to 100%). The error bars represent standard error of the mean.
Figure 2
DSS1 silencing confers chromosomal sensitivity to mitomycin C in mammalian cells. Embryonic stem cells, transfected with pSUPER–NS or pSUPER–mDss1, were treated with mitomycin C (0.2 μg/ml) 48 h after transfection and chromosomes were harvested the next day. Arrows in panels (A) and (B) indicate chromatid/chromosome breaks and complex chromosome rearrangements, respectively.
Figure 3
DSS1 is required for RAD51 focus formation after DNA damage. (A) Embryonic stem (ES) cells and MCF7 cells were transfected with pSUPER–NS or pSUPER–mDss1/pSUPER–hDSS1, respectively. After 24 h, the cells were irradiated and 5 h later were fixed and stained for RAD51 foci as described by Tarsounas et al (2003). Nuclei are shown in blue and RAD51 foci in red. (B) The percentage of cells with >5 foci was quantified. The error bars represent s.e.m.
Figure 4
DSS1 is not required for BRCA2 and RAD51 stability or the interaction of BRCA2 and RAD51. Embryonic stem (ES) cells were transfected with either pSUPER–eGFP–NS or pSUPER–eGFP–mDss1 and expression levels of myc-tagged Brca2 and Rad51 were analysed after 24 h by western blotting. Blots were probed with either anti-c-myc (A-14) antibody for Brca2 levels or anti-Rad51 Ab-1 (3C10) antibody. Blots were stripped and re-probed with anti-β-tubulin antibody as a loading control. To analyse the interaction between Brca2 and Rad51, lysates were incubated with protein G–Sepharose beads and with c-myc antibody (Brca2) or anti-GFP antiserum (mock) and bound proteins were analysed by western blotting. Lysate from ES cells with untagged Brca2 was used as a control for the immunoprecipitation. Blots were probed with anti-Rad51 antibody. Each blot also shows a titration of lysate from untransfected ES cells to demonstrate the detection level.
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