Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells

Abstract

Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or Rad51C. Ni2+-binding experiments demonstrate that XRCC3 and Rad51C interact in human cells. In addition, we find that Rad51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2), both containing Rad51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of Rad51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize Rad51C.

Figure 1

Expression levels of recombinant proteins in stable transfectants of human cell lines. Western blot analysis of 25 µg of protein extracted from each cell line was used to monitor the protein expression levels of recombinant XRCC3-(His)₆ in TK6-XRCC3 cells and Rad51C-(His)₆ in WTK1-Rad51C cells. (A) Native and recombinant XRCC3 was detected using polyclonal anti-XRCC3 antibody (P. Sung). (B) Native and recombinant Rad51C were detected using polyclonal anti-Rad51C antibody (P. Sung).

Figure 2

Constitutive and X-ray-induced levels of the Rad51 paralogs in different human cell lines. Western blot analysis using different antibodies was used to determine the constitutive level of the Rad51 paralogs and of hRad51, and the level of these proteins 4 and 8 h after treatment with 10 Gy X-rays. P53 was used as a control for the X-ray treatment (see text), and either β-tubulin or transcription factor QM was used as loading standards for each western blot (only one representative blot for each is shown). Unlike Figure 1, the detection of XRCC3 here was done using an antibody from Novus that only weakly recognizes the recombinant XRCC3-(His)₆ protein (see text).

Figure 3

Interactions of XRCC3 and Rad51C, and of Rad51C with Rad51B, Rad51D and XRCC2 in human cells. Following Ni²⁺ pull down of recombinant Rad51C-(His)₆ and XRCC3-(His)₆ from untreated cells, western blot analysis was used to determine which of the Rad51 paralogs interacted constitutively. Rad51C-(His)₆ is directly pulled down in the first lane, and XRCC3-(His)₆ is directly pulled down in the third lane. WTK1 and TK6 (second and fourth lanes) are negative controls. (A) Western blot using antibody against Rad51C (from P. Sung). (B) Western blot using antibody against XRCC3 (P. Sung). (C–E) Western blots using antibodies against Rad51B (J. Albala), Rad51D (Novus) and XRCC2 (P. Sung), respectively, showing that these proteins are associated with Rad51C-(His)₆, but not with XRCC3-(His)₆. (F) Western blots using antibody against Rad51 (Calbiochem). A small fraction of native Rad51C, XRCC3 and Rad51 were non-specifically bound to the Ni²⁺ beads (A, B and F).

Figure 4

Interactions between Rad51 paralogs in Ni²⁺ pull-down experiments are unaffected by DNA damage. (A) WTK1-Rad51C and TK6-XRCC3 cells were treated with 10 Gy X-ray and protein extracts (same as used in Fig. 2) were used in Ni²⁺ pull-down experiments. In the first three lanes, Rad51C-(His)₆ is pulled down, and in the last three lanes, XRCC3-(His)₆ is pulled down. (B) Treatment of TK6-XRCC3 with MMC (0.5 µg/ml for 4 h) does not affect the interaction of Rad51C with XRCC3-(His)₆. In the first lane, Rad51C-(His)₆ is pulled down, and in the last two lanes, XRCC3-(His)₆ is pulled down. TK6 was the negative control in the top panel and WTK1 was used as a negative control in the bottom panel (second lane in each case). Please note that the upper part of (B) was probed with a monoclonal antibody against Rad51C, and this antibody does not recognize the recombinant form of Rad51C-(His)₆. This is very probably due to the interference of the His₆-tag with the C-terminally directed epitope of this antibody.

Figure 5

Immunoprecipitation of native Rad51C from TK6 cells results in co-immunoprecipitation of the other four Rad51 paralogs, but not Rad51. A monoclonal Rad51C antibody was used for immunoprecipitation (right hand lanes), and normal mouse IgG was used as a negative control (left hand lanes). (A–F) Polyclonal antibodies specific for each Rad51 paralog and for Rad51 were used in western detection.

Figure 6

Summary of interactions observed between Rad51 paralogs in human cells. (A) Evidence from this study suggests that human cells contain two different complexes of Rad51 paralogs (upper row), but our data could also be explained by three different complexes (lower row) or a mixture of these complexes. (B) One large complex containing all of the Rad51 paralogs seems unlikely to exist in human cells because XRCC3 does not appear to associate with Rad51B, Rad51D or XRCC2.