Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells

Abstract

Genetic studies in rodent and chicken mutant cell lines have suggested that Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3) play important roles in homologous recombinational repair of DNA double-strand breaks and in maintaining chromosome stability. Previous studies using yeast two- and three-hybrid systems have shown interactions among these proteins, but it is not clear whether these interactions occur simultaneously or sequentially in vivo. By utilizing immunoprecipitation with extracts of human cells expressing epitope-tagged Rad51 paralogs, we demonstrate that XRCC2 and Rad51D, while stably interacting with each other, co-precipitate with Rad51C but not with XRCC3. In contrast, Rad51C is pulled down with XRCC3, whereas XRCC2 and Rad51D are not. In addition, Rad51B could be pulled down with Rad51C and Rad51D, but not with XRCC3. These results suggest that Rad51C is involved in two distinct in vivo complexes: Rad51B-Rad51C-Rad51D-XRCC2 and Rad51C-XRCC3. In addition, we demonstrate that Rad51 co-precipitates with XRCC3 but not with XRCC2 or Rad51D, suggesting that Rad51 can be present in an XRCC3-Rad51C-Rad51 complex. These complexes may act as functional units and serve accessory roles for Rad51 in the presynapsis stage of homologous recombinational repair.

Figure 1

Expression of HA-Rad51D in human cells and interaction of XRCC2 with HA-Rad51D. (A) Western blots with α-HA antibody of cell extracts from HeLaS3 subclones transfected with HA-Rad51D expression vector. Clones HD19 and HD25 expressed HA-Rad51D at a molecular weight at ∼36 kDa while clones HD16 and HD21 show little or no expression. (B) Immunoprecipitation of HA-Rad51D with HA affinity matrix in HD16 and HD19 extracts with or without irradiation. HA-Rad51D was pulled down in HD19 and detected on western blot with HA antibody. Native XRCC2 was specifically detected in the HA-Rad51D precipitates using either rabbit XRCC2 (α-XRCC2r) or mouse XRCC2 (α-XRCC2m) antibody. (C) HA-Rad51D was immunoprecipitated in HD19 extracts using HA affinity matrix at increasing concentrations of NaCl. XRCC2 was co-precipitated under all the conditions tested.

Figure 2

Co-immunoprecipitation of Rad51D, Rad51C and XRCC2. (A) Immunoprecipitation of HD16 and HD19 extracts with α-XRCC2 (Novus, lane 1), α-Rad51D (Novus, lane 2) and α-HA (lanes 3 and 4). The western blot was probed with α-Rad51D. The native Rad51D and HA-Rad51D are indicated. The dark band at ∼55 kDa (lanes 1 and 2) is the heavy chain of rabbit IgG [the same in (B)]. (B) Immunoprecipitation of HD16 and HD19 extracts with α-XRCC2 (Novus, lane 1) and α-HA (lanes 2 and 3). The XRCC2 (top) and Rad51C (bottom) were detected using α-XRCC2r and α-Rad51C antibody, respectively. Rad51C co-precipitates with XRCC2 (lane 1) or HA-Rad51D (lane 3).

Figure 3

Interaction of XRCC3 with Rad51C but not with Rad51D and XRCC2. The cells were incubated for 2 h after 8 Gy γ-irradiation. (A) Immunoprecipitation of control HeLa and H158 (expressing HA-XRCC3) cell extracts with HA affinity matrix. The western blots were incubated with α-HA (top) or α-HsRad51 (bottom). HA-XRCC3 is detected in H158 cells but not in the untransfected control, and Rad51 co-precipitates with HA-XRCC3. (B) Immunoprecipitation analysis in H158 and HD19 (expressing HA-Rad51D) was done using HA affinity matrix. The western blots were incubated with: a, α-Rad51D; b, α-XRCC3; c, α-Rad51C; and d, α-XRCC2r.

Figure 4

Association of Rad51B with Rad51D and Rad51C. Western blot was performed with the antibody indicated at the top of each panel. (A) Rad51B was co-precipitated with Rad51C (lanes 1 and 2). Rad51B antibodies from Novus (lane 1) or from Dr Patrick Sung (lane 2) were used for immunoprecipitation. Lane 3 is a control of Rad51C that was pulled down by α-Rad51C. (B) Rad51B was not co-precipitated with XRCC3 (lane 2), while it was co-precipitated with Rad51C (lane 1) and Rad51D (lane 3). (C) Lane 1, Rad51D was co-precipitated with Rad51B (Novus antibody). Lanes 2 and 3 are controls showing the position of Rad51D, which is pulled down with α-Rad51D or α-Rad51C.

Figure 5

Lack of association of XRCC2 with Rad51. (A) Immunoprecipitation with HA affinity matrix in HD16 and HD19 cell extracts. The blots were incubated with α-HA (top) or α-HsRad51 (bottom). (B) Immunoprecipitation with Flag affinity matrix in wild type, irs1 and Flag-XRCC2 transformants of irs1 (IFX200). The blots were incubated with α-XRCC2r (top) or α-MmRad51 (bottom). (C) Immunofluorescence detection of GFP-XRCC2 in stable transformants of wild-type V79 cells 2 h after 0 or 8 Gy γ-irradiation (top). The same cells stained with DAPI (bottom).

Figure 6

Speculative model of complexes and dynamic interactions among Rad51 paralogs and HsRad51. The diagram takes into account the results by Braybrooke and co-workers showing an XRCC2–Rad51D dimer in cell extracts (31) and the data from yeast two-hybrid analyses showing that Rad51B and Rad51C can form a dimer (30). XRCC2–Rad51D and Rad51B–Rad51C dimers combine to form the Rad51B–C–D–XRCC2 complex. XRCC3 may promote the dissociation of Rad51C from this complex to produce the XRCC3–Rad51C dimer that binds to Rad51. A possible conformational change as depicted for Rad51C could contribute to the specificity of interactions.