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. 2001 Jul 17;98(15):8440-6.
doi: 10.1073/pnas.111005698.

Complex formation by the human RAD51C and XRCC3 recombination repair proteins

J Y Masson  1 , A Z Stasiak, A Stasiak, F E Benson, S C West
Affiliations

Complex formation by the human RAD51C and XRCC3 recombination repair proteins

J Y Masson et al. Proc Natl Acad Sci U S A. .

Abstract

In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C--XRCC3 complex binds single-stranded, but not duplex DNA, to form protein--DNA networks that have been visualized by electron microscopy.

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Figures

Figure 1
Figure 1
Copurification of RAD51C and XRCC3 from baculovirus-infected insect cells. (A) Purification of RAD51C-His10 and XRCC3-His6. Lane a, molecular weight markers. Lanes b–j, elution profile from the Talon column. Proteins were visualized by SDS/PAGE followed by Coomassie blue staining. (B) Western blot of purified RAD51C-His10 and XRCC3-His6 using mAbs raised against RAD51C or XRCC3 (mAbs 2H11 and 10F1, respectively).
Figure 2
Figure 2
RAD51C forms a stable complex with XRCC3 in insect cells. (A) Coimmunoprecipitation of RAD51C with XRCC3. Sf9 cells were coinfected with RAD51C-His10 and XRCC3-His6 baculovirus. Immunoprecipitations were carried out as described in Materials and Methods by using preimmune serum (lane b) or a pAb raised against RAD51C (lanes c–f). Complexes were washed in lysis buffer with the indicated concentration of NaCl, and visualized by Western blotting using an anti-histidine mAb. Lane a, purified RAD51C-His10–XRCC3-His6 complex (50 ng). (B) Specificity of the anti-RAD51C pAb. Sf9 cells were infected with an XRCC3-His6 baculovirus, and cell-free extracts were prepared in lysis buffer. Lane a, control showing purified RAD51C-His10–XRCC3-His6 complex (50 ng). Lanes b–e, pull downs by using preimmune RAD51C serum (lane b), anti-RAD51C pAb (lane c), preimmune XRCC3 serum (lane d), or anti-XRCC3 pAb (lane e). The immunoprecipitates were analyzed by Western blotting using an anti-histidine mAb and ECL.
Figure 3
Figure 3
Coimmunoprecipitation of endogenous RAD51C and XRCC3 from HeLa cell-free extracts. Protein complexes were precipitated from HeLa cell-free extracts by using preimmune serum (lanes b and h) or pAbs raised against RAD51C (lanes c–f and i–l). The complexes were washed in buffer containing NaCl, as indicated, and visualized by Western blotting using anti-RAD51C (lanes a–f) or anti-XRCC3 mAbs (lanes g–l). Lanes a and g, purified RAD51C-His10–XRCC3-His6 complex (50 ng). The His-tagged controls migrate more slowly than the endogenous RAD51C or XRCC3 from HeLa.
Figure 4
Figure 4
Visualization of RAD51C–XRCC3 by electron microscopy. (A) RAD51C–XRCC3 complex. (B) Human DMC1 protein. The white arrow indicates a RAD51C–XRCC3 structure containing a cavity. The bar represents 50 nm.
Figure 5
Figure 5
DNA binding by RAD51C–XRCC3. (A) Reactions (10 μl) contained single-stranded (lanes a–f), double-stranded (lanes g–l), 5′-tailed (lanes m–r), or 3′-tailed (lanes s–y) DNA in binding buffer, and the indicated amounts of RAD51C–XRCC3. Protein–DNA complexes were analyzed by PAGE. 5′-32P-end labels are indicated with asterisks. (B) The gels shown in A were quantified by phosphorimaging.
Figure 6
Figure 6
Requirements for RAD51C–XRCC3-mediated DNA aggregation. DNA binding reactions were carried out as described in Fig. 5 by using binding buffer modified as indicated. In lanes i–m, the concentration of RAD51C–XRCC3 was 20 nM.
Figure 7
Figure 7
Electron microscopic visualization of complexes formed between RAD51C–XRCC3 and DNA. Reactions contained RAD51C–XRCC3, with pPB4.3-gapped circular DNA (A), pPB4.3-tailed linear DNA (B), pDEA-7Z linear duplex DNA (C), or pPB4.3-supercoiled DNA (D). The bar represents 100 nm.
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