The homologous-pairing activities of the RAD51A1(A2L2) and RAD51A2(A1L2) mutants.

<p>(A) Purified RAD51A1(A2L2) and RAD51A2(A1L2) mutants. Lane 1 indicates the molecular mass markers, and lanes 2 and 3 represent RAD51A1 and RAD51A2, respectively. Lanes 4 and 5 represent RAD51A1(A2L2) and RAD51A2(A1L2). An aliquot (0.5 µg) of each protein was analyzed. (B) The D-loop formation assay (Protein titration experiments). The indicated amounts of rice RAD51A1, RAD51A2, RAD51A1(A2L2), or RAD51A2(A1L2) were incubated with the ³²P-labeled 50-mer ssDNA, and the homologous-pairing reaction was initiated by the addition of superhelical dsDNA. Reactions were allowed to proceed for 5 min. Lane 1 indicates a negative control experiment without protein, and lanes 2–5, 6–9, 10–13, and 14–17 represent the reactions conducted with RAD51A1, RAD51A2, RAD51A1(A2L2), and RAD51A2(A1L2), respectively. The protein concentrations were 0.4 µM (lanes 2, 6, 10, and 14), 0.6 µM (lanes 3, 7, 11, and 15), 0.9 µM (lanes 4, 8, 12, and 16), and 1.2 µM (lanes 5, 9, 13, and 17).</p

Purification of rice RAD51A1 and RAD51A2.

<p>(A) The amino acid sequences of rice RAD51A1 and RAD51A2 from japonica cultivar group, cv. Nipponbare, rice RAD51 from indica cultivar group, cv. Pusa Basmati 1, and human RAD51, aligned with the ClustalX software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075451#pone.0075451-Thompson1" target="_blank">[50]</a>. Black and gray boxes indicate identical and similar amino acid residues, respectively. The L1 and L2 loops, which are important for DNA binding, are represented by red lines. (B) Purified rice RAD51A1, RAD51A2, and human RAD51. Lane 1 indicates the molecular mass markers, and lanes 2, 3, and 4 represent rice RAD51A1 (0.5 µg), RAD51A2 (0.5 µg), and human RAD51 (0.5 µg), respectively. (C) The ATPase activities of <i>Oryza sativa</i> RAD51A1 and RAD51A2. The reactions were conducted with φX174 circular ssDNA (left panel), linearized φX174 dsDNA (center panel), or without DNA (right panel), in the presence of 5 µM ATP. Blue circles and red squares represent the experiments with RAD51A1 and RAD51A2, respectively. The averages of three independent experiments are shown with the SD values.</p

Interactions of rice RAD51A1 and RAD51A2.

<p>(A) Purified His₆-tagged RAD51A1 protein. Lane 1 indicates the molecular mass markers, and lanes 2 and 3 represent the RAD51A1 and His₆-tagged RAD51A1 proteins (0.5 µg). (B) The pull-down assay with Ni–NTA beads. Lane 1 represents molecular mass markers. Lanes 2, 3, and 4 show purified protein controls of His₆-tagged RAD51A1, RAD51A1, and RAD51A2, respectively. Lane 5 indicates a negative control experiment without RAD51A1 and RAD51A2, in the presence of His₆-tagged RAD51A1. Lanes 8 and 9 indicate negative control experiments with RAD51A1 and RAD51A2, respectively, in the absence of His₆-tagged RAD51A1. Lanes 6 and 7 indicate experiments with RAD51A1 and RAD51A2, respectively, in the presence of His₆-tagged RAD51A1. The proteins bound to His₆-tagged RAD51A1 were pulled down by the Ni–NTA agarose beads. The samples were fractionated by 10% SDS–PAGE, and the protein bands were visualized by Coomassie Brilliant Blue staining. (C) The D-loop formation assay in the presence of Ca²⁺(Protein titration experiments). The indicated amounts of rice RAD51A1 and RAD51A2 were mixed and incubated with the ³²P-labeled 50-mer ssDNA, and the homologous-pairing reaction was initiated by the addition of superhelical dsDNA. Reactions were allowed to proceed for 5 min. Lane 1 indicates a negative control experiment without protein, and lanes 2–4 and 10–12 indicate positive control experiments with RAD51A1 and RAD51A2, respectively. The protein concentrations were 0.2 µM (lanes 2 and 12), 0.4 µM (lanes 3 and 11), and 0.6 µM (lanes 4 and 10). Lanes 5–9 represent experiments with various amounts of RAD51A1 and RAD51A2. Lane 5: RAD51A1 (0 µM) and RAD51A2 (0.6 µM). Lane 6: RAD51A1 (0.2 µM) and RAD51A2 (0.4 µM). Lane 7: RAD51A1 (0.3 µM) and RAD51A2 (0.3 µM). Lane 8: RAD51A1 (0.4 µM) and RAD51A2 (0.2 µM). Lane 9: RAD51A1 (0.6 µM) and RAD51A2 (0 µM).</p

The polymerization activities of rice RAD51A1 and RAD51A2.

<p>A 70 µg portion of RAD51A1 (A) or RAD51A2 (B) was analyzed by Superdex 200 gel filtration chromatography. The top, middle, and bottom panels represent the experiments without ATP, with ATP, and with ADP, respectively.</p

Electron microscopic images of RAD51A1 and RAD51A2 complexed with DNA.

<p>(A and B) Electron microscopic images of rice RAD51A1 (A) and RAD51A2 (B) filaments formed on the φX174 dsDNA in the presence of ATP. The average helical pitches of the RAD51A1 and RAD51A2 filaments were about 9.15 nm. The black bar denotes 100 nm.</p

The DNA-binding activities of rice RAD51A1 and RAD51A2.

<p>Circular φX174 ssDNA (20 µM) (A) or linear φX174 dsDNA (20 µM) (C) was incubated with rice RAD51A1, RAD51A2, or human RAD51 at 37°C for 10 min. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer, and were visualized by ethidium bromide staining. Lanes 1–10 and 11–20 represent the reactions conducted with and without ATP, respectively. Lanes 1 and 11 indicate negative control experiments without protein. Lanes 2–4 and 12–14 represent the experiments conducted with RAD51A1. Lanes 5–7 and 15–17 represent the experiments conducted with RAD51A2. Lanes 8–10 and 18–20 represent the experiments conducted with human RAD51. The protein concentrations were 0.75 µM (lanes 2, 5, 8, 12, 15, and 18), 1.5 µM (lanes 3, 6, 9, 13, 16, and 19) and 3 µM (lanes 4, 7, 10, 14, 17, and 20). (B) Graphic representation of the relative migration distances of the RAD51A1- and RAD51A2-ssDNA complexes. The migration distances relative to the free DNA are plotted against the protein concentrations. (D) Competitive ssDNA- and dsDNA-binding. Circular φX174 ssDNA (20 µM) and linear φX174 dsDNA (20 µM) were incubated with rice RAD51A1, RAD51A2, or human RAD51 at 37°C for 10 min, under the 120 mM NaCl conditions. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer, and were visualized by ethidium bromide staining. Lane 1 indicates negative control experiments without protein. Lanes 2–4, 5–7, and 8–10 represent the experiments conducted with RAD51A1, RAD51A2, and human RAD51, respectively. The protein concentrations were 0.9 µM (lanes 2, 5, and 8), 1.8 µM (lanes 3, 6, and 9), and 3.6 µM (lanes 4, 7, and 10).</p

The homologous pairing activities of rice RAD51A1 and RAD51A2.

<p>(A) A schematic representation of the D-loop formation assay. Asterisks indicate the ³²P-labeled end of the 50-mer ssDNA. (B) The D-loop formation assay in the presence of Ca²⁺(Protein titration experiments). The indicated amounts of rice RAD51A1, RAD51A2, or human RAD51 were incubated with the ³²P-labeled 50-mer ssDNA, and the homologous-pairing reaction was initiated by the addition of superhelical dsDNA. Reactions were allowed to proceed for 5 min. Lane 1 indicates a negative control experiment without protein, and lanes 2–6, 7–11, and 12–16 represent the reactions conducted with RAD51A1, RAD51A2, and human RAD51, respectively. The protein concentrations were 0.2 µM (lanes 2, 7, and 12), 0.4 µM (lanes 3, 8, and 13), 0.6 µM (lanes 4, 9, and 14), 0.9 µM (lanes 5, 10, and 15), and 1.2 µM (lanes 6, 11, and 16). (C) Graphic representation of the experiments shown in panel B. The averages of three independent experiments are shown with the SD values. Blue circles, red squares, and green triangles represent the experiments with RAD51A1, RAD51A2, and human RAD51, respectively. (D) The D-loop formation assay without Ca²⁺(Protein titration experiments). The homologous pairing reactions were conducted without Ca²⁺, and were performed according to the same procedure as shown in panel B. (E) Graphic representation of the experiments shown in panel D. The averages of three independent experiments are shown with the SD values. Blue circles, red squares, and green triangles represent the experiments with RAD51A1, RAD51A2, and human RAD51, respectively. (F) Graphic representation of the D-loop formation assay (time course experiments). The homologous pairing reactions were conducted according to the same procedure as shown in panel B. Rice RAD51A1 (0.5 µM) or RAD51A2 (0.5 µM) was used in the time course experiments.</p