Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy

Abstract

In mammals, there are five Rad51 paralogs that form two distinct complexes in vivo. One complex is composed of Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and the other Rad51C-Xrcc3 (CX3). We co-expressed and purified human BCDX2 and CX3 protein complexes from insect cells and investigated their binding preferences and structure using transmission electron microscopy (TEM). We visualized the binding of BCDX2 and CX3 to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both complexes bind with exceptionally high specificity to the DNA junctions with little binding observed elsewhere on the DNAs. Further analysis of the structure of free or DNA-bound BCDX2 and CX3 complexes revealed a multimeric ring structure whose subunits are arranged into a flat disc around a central channel. This work provides the first EM visualization of BCDX2 and CX3 binding to Holliday junctions and forked DNAs and suggests the complexes form ring-shaped structures.

FIGURE 1.

Purification of human BCDX2 and CX3 Rad51 paralog complexes. The His-tagged human Rad51B, Rad51C, Rad51D, and Xrcc2 or Rad51C and Xrcc3 complexes were purified from multiply infected Sf21 insect cells as described under “Experimental Procedures.” Protein complexes were separated on a 10% SDS-PAGE gel and visualized by Coomassie Blue staining. A, purified complex containing Rad51B, Rad51C, Rad51D, Xrcc2. B, purified complex containing Rad51C and Xrcc3.

FIGURE 2.

BCDX2 and CX3 binding to replication fork DNAs as seen by EM. Replication fork DNAs were incubated with A, BCDX2 or B, CX3, and the products were visualized by EM. Samples were mounted onto charged carbon-coated copper grids and shadowed with tungsten. Representative bound replication forks are shown. The bar is equivalent to 50 nm. C, CX3 and D, BCDX2 protein complexes at higher magnification. The bar is equivalent to 9 nm.

FIGURE 3.

Quantitative analysis of BCDX2 and CX3 binding to replication forks and Holliday junctions. Binding was characterized as either unbound or bound and expressed as a percentage of molecules analyzed. A, the percentage of replication forks bound by BCDX2 (n = 162) or CX3 (n = 210). C, the percentage of Holliday junctions bound by BCDX2 (n = 258) or CX3 (n = 182). The percentage of bound molecules was further categorized as either bound at the junction or bound elsewhere (tail/other) on the DNA and expressed as the percentage of total bound molecules. B, percentage of bound replication forks with BCDX2 or CX3 at the junction. D, percentage of bound Holliday junctions with BCDX2 and CX3 at the junction. Error bars represent ± S.D.

FIGURE 4.

Quantitative analysis of BCDX2 and CX3 bound replication forks containing different amounts of ssDNA at the fork junction. Large numbers of DNA molecules were surveyed to determine protein-free and protein-bound fractions on replication forks containing 1, 5, 15, or 25 nt of ssDNA at the junction of the fork. A, replication forks containing 1-, 5-, 15-, or 25-nt gaps bound by BCDX2 or B, CX3 were examined from two-three independent experiments. Molecules were scored as unbound, bound at base of the three-way forked junction, or bound nonspecifically (defined as along the arm or along the circular portion of the template). Error bars are ± S.D. C and D, the protein-bound fractions were scored for specific junction binding and nonspecific binding (defined as binding elsewhere on the DNA) for BCDX2 and CX3, respectively. Error bars represent ± S.D.

FIGURE 5.

Binding of BCDX2 or CX3 to Holliday Junction DNA. CX3 (A) or BCDX2 (B) was incubated with Holliday junction templates, mounted onto carbon-coated copper grids, and rotary shadowcast with tungsten for visualization by EM. Images are shown in reverse contrast. The bar is equivalent to 50 nm.

FIGURE 6.

Visualization of BCDX2 and CX3 ring-shaped complexes by negative staining electron microscopy. A, representative elution profile of BCDX2 complex from Superdex 200 column. The major peak contained all four Rad51 paralogs. Arrows indicate elution of thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa). B, representative field of BCDX2 complexes visualized by mounting the peak fraction onto a charged carbon grids followed by staining with 2% uranyl acetate as described under “Experimental Procedures.” The bar is 100 nm. C, negatively stained BCDX2 fractions with rings of mixed size diameter. The bar is 50 nm. D and E, panel of rings observed by negative staining of the BCDX2 and CX3 fractions, respectively. The bar is 50 nm.