Identification and purification of two distinct complexes containing the five RAD51 paralogs - PubMed (original) (raw)
Identification and purification of two distinct complexes containing the five RAD51 paralogs
J Y Masson et al. Genes Dev. 2001.
Abstract
Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.
Figures
Figure 1
Interactions between the RAD51 paralogs in HeLa cell-free extracts. Coimmunoprecipitation of endogenous RAD51B, RAD51C, RAD51D, and XRCC2 from HeLa cell-free extracts. Protein complexes were precipitated using preimmune serum (PI; lane b) or anti-RAD51D pAbs (lanes c_–_f). The complexes were washed in buffer containing NaCl, as indicated, and visualized by Western blotting using anti-RAD51B, anti-RAD51C, anti-RAD51D, anti-XRCC2, or anti-XRCC3 mAbs, as indicated. (Lane a) Marker proteins (RAD51Bhis6, RAD51Chis10, RAD51Dhis6, XRCC2, XRCC3his6). The His-tagged controls migrate more slowly than the endogenous human proteins.
Figure 2
Specific interactions between XRCC3 and RAD51C. Sf9 cells were coinfected with pBAC51B, pBAC51C, pBAC51D, pBACX2, and pBACX3his6. Following protein expression, extracts were loaded onto a Talon column to bind XRCC3-His6 and associated proteins, which were subsequently eluted and identified by Western blotting using mAbs. (Lane a) Flow through; (lane b) 30 mM imidazole wash; (lanes c_–_j) fractions 4–18.
Figure 3
Complex formation by the RAD51 paralogs. (A) Gel filtration analysis of RAD51 paralog complexes. Extracts from Sf9 cells infected with pBAC51B, pBAC51Chis10, pBAC51D, pBACX2, and pBACX3his6 were analyzed by gel filtration as described in Materials and Methods. (B) Diagrammatic representation of two distinct complexes as indicated by the data presented in Figures 1 and 2 and part A.
Figure 4
Dissection of the interactions between RAD51C, RAD51B, RAD51D, and XRCC2. Sf9 cells were infected with the indicated baculovirus and extracts were loaded onto a Talon column to bind the single His-tagged protein and associated proteins. The interacting proteins were eluted and identified by Western blotting using mAbs. The column flow through (FT), wash (W), and eluting fractions (4–20) are indicated. A summary of interactions is shown diagrammatically at the right. (A) Sf9 infected with pBAC51Chis10 and pBAC51B. (B) pBAC51Chis10 with pBAC51D. (C) pBAC51Chis10 expressed with both pBAC51B and pBAC51D. Blot probed with RAD51C mAbs (top panel), and with RAD51B and RAD51D mAbs (lower panel). (D) pBAC51Bhis6 with pBAC51D. (E) pBACX2his6 with both pBAC51B and pBAC51C. Blot probed with RAD51B and XRCC2 mAbs (upper panel), and with RAD51C and XRCC2 mAbs (lower panel).
Figure 5
Purification of two distinct complexes containing the RAD51 paralogs from baculovirus-infected insect cells. (A) Purification of a complex containing RAD51B, RAD51C, RAD51D, and XRCC2 (BCDX2). The complex was purified from Sf9 cells infected with pBAC51Bhis6, pBAC51Chis10, pBAC51D, and pBACX2, as described in Materials and Methods. Proteins were visualized by SDS-PAGE followed by Coomassie blue staining. (Lane a) Molecular mass markers; (lanes b,c) 2.0 and 3.5 μg of protein. (B) Western blot of purified BCDX2 complex using mAbs raised against RAD51B, RAD51C, RAD51D, and XRCC2. (C) Purified RAD51C–XRCC3 complex. Proteins were purified from Sf9 cells infected with pBAC51Chis10 and pBACX3his6 and visualized by SDS-PAGE followed by Coomassie blue staining. (Lane a) Molecular mass markers; (lane b) 3 μg of RAD51C–XRCC3 complex.
Figure 6
DNA-binding and DNA-stimulated ATPase activities of BCDX2 complex. (A) DNA-binding reactions containing ssDNA, dsDNA, or tailed duplex DNA and the indicated concentrations of BCDX2 were carried out as described in Materials and Methods. Protein–DNA complexes were analyzed by PAGE. 5′-32P-end labels are indicated with asterisks. (B) Quantification of the data shown in A by PhosphorImaging. (C) The ATPase activities of the BCDX2 complex were analyzed in the presence and absence of DNA, as indicated.
Figure 7
Interaction of BCDX2 complex with single-stranded regions in duplex DNA. (A_–_C) Electron microscopic visualization of complexes formed between BCDX2 and tailed duplex DNA (A) or gapped circular duplex DNA (B). The bound single-stranded regions are indicated by white arrows. (C) Control indicating the failure of BCDX2 to bind linear duplex DNA. (D) Juxtaposition of BCDX2 complex and RAD51 on gapped circular DNA. The DNA was preincubated with BCDX2 complex (90 nM) for 5 min and then supplemented with RAD51 (0.3 μM). After 5 min at 37°C, the products were visualized by electron microscopy. The white arrow indicates a RAD51 filament, and the black arrow indicates the BCDX2–DNA complexes.
Figure 8
Interaction of BCDX2 complex with nicks in duplex DNA. Binding reactions contained linear duplex, nicked linear duplex, or nicked circular plasmid DNA, as indicated. Protein–DNA complexes were analyzed by agarose gel electrophoresis (A) or by electron microscopy (B,C). White arrows indicate the binding of BCDX2 to the single nick present in each DNA substrate. The bar represents 100 nm.
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