FIGURE 4.

SWSAP1 interacts with RAD51 and RAD51 paralogs and is involved in homologous recombination repair. a, cells with hSWS1 or SWSAP1 down-regulation display increased MMS but not CPT sensitivity. Cell survival assays were performed as described under “Experimental Procedures.” Data are presented as means ± S.D. (error bars) from three different experiments. b, schematic representation of HR assay. The DR-GFP construct consists of direct repeats of two mutated GFP genes, SceGFP and the truncated iGFP. When a single double strand DNA break generated by I-Sce1 is repaired via gene conversion with iGFP, expression of GFP is restored and can be measured by FACS analysis. c, decreasing hSWS1 or SWSAP1 expression impairs HR repair. U2OS DR-GFP cells were electroporated with pCBASce plasmids (see “Experimental Procedures” for details). The percentage of GFP-positive cells was determined by flow cytometry 48 h after electroporation. The data were normalized to those obtained from cells transfected with control siRNA (set as 1.0). Means ± S.E. (error bars) shown are obtained from three independent experiments. d and e, down-regulation of hSWS1 or SWSAP1 impairs MMS-induced RAD51 foci formation. Immunostaining was performed 6 h after MMS treatment using the indicated antibodies. Representative RAD51 foci are shown in d. Data were presented as means ± S.D. (error bars) from three different experiments, more than 100 cells were counted in each experiment (e). f, knockdown efficiency was confirmed by immunoblotting. g, SWSAP1 interacts with RAD51 and several RAD51 paralogs, but not with XRCC2 in vivo. 293T cells were transfected with plasmids encoding SFB-tagged SWSAP1 together with plasmids encoding Myc-tagged RAD51 or RAD51 paralogs. Cells were collected 24 h after transfection. Precipitation reactions were performed using S beads, and immunoblotting was carried out using antibodies as indicated. h, SWSAP1 interacts with RAD51 and several RAD51 paralogs, but not with XRCC2 in vitro. SF9 cells were co-infected with baculoviruses expressing GST-tagged RAD51 or RAD51 paralogs together with those expressing SFB-tagged SWSAP1. Pulldown experiments and immunoblotting were carried out as indicated. i, the ATPase activity of SWSAP1 is required for restoring cellular resistance to MMS. HeLa-derivative cell lines stably expressing siRNA-resistant HA-FLAG-tagged WT (SWSAP1SiR-WT), K18A mutant (SWSAP1SiR-KA), and D96A mutant (SWSAP1SiR-DA) of SWSAP1 were generated. Cell survival assays were performed as described under “Experimental Procedures.” Data are presented as means ± S.D. (error bars) from three different experiments. j, the ATPase activity of SWSAP1 is required for its function in promoting RAD51 foci formation. Immunostaining was performed 6 h after MMS treatment using indicated antibodies. Data are presented as means ± S.D. (error bars) from three different experiments, more than 100 cells were counted in each experiment. k, SWSAP1 expression was confirmed by immunoblotting with the use of FLAG antibody, and extracts were prepared from cells transfected with SWSAP1 siRNA#2.