Homologous-pairing activity of the human DNA-repair proteins Xrcc3.Rad51C

Abstract

The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C/Rad51L2. The purified Xrcc3.Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3.Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3.Rad51C. Electron microscopic observations revealed that Xrcc3.Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.

Figures

Figure 1

The purified Xrcc3⋅Rad51C and Rad51C proteins. (A) SDS/12% PAGE of Ni column fractions of Xrcc3⋅Rad51C. Fractions (3 μl) containing Xrcc3⋅Rad51C were analyzed by SDS/12% PAGE. Fraction numbers are indicated at the top of the gel. (B) SDS/12% PAGE of the purified Xrcc3⋅Rad51C and Rad51C proteins. Lane 2 is Xrcc3⋅Rad51C (0.5 μg) eluted from an Affi-Gel-heparin column, and lane 3 is Rad51C (0.5 μg) eluted from Ni nitrilotriacetate agarose. Lane 1 is molecular mass markers. The proteins were visualized by Coomassie brilliant blue staining. (C) Elution profiles of Xrcc3⋅Rad51C and HsRad51 in Superdex 200 h chromatography. Xrcc3⋅Rad51C (20 μg) and HsRad51 (20 μg) were analyzed by Superdex 200 h column chromatography. (Upper) Elution profile of HsRad51. (Lower) Elution profile of Xrcc3⋅Rad51C.

Figure 2

DNA-binding activity of Xrcc3⋅Rad51C. (A) The ssDNA binding of Xrcc3⋅Rad51C. A 32P-labeled single-stranded 50-mer oligonucleotide (100 nM), which does not contain intramolecular base pairing, was incubated with Xrcc3⋅Rad51C at 37°C for 10 min, and the reactions were analyzed by nondenaturing 8% PAGE in TBE buffer. Concentrations of Xrcc3⋅Rad51C were 55 nM (lane 2), 110 nM (lane 3), and 220 nM (lane 4) and were calculated as heterodimers. (B) The dsDNA binding of Xrcc3⋅Rad51C. A 32P-labeled double-stranded 50-mer oligonucleotide (200 nM) was used as the substrate. Concentrations of Xrcc3⋅Rad51C were 55 nM (lane 2), 110 nM (lane 3), and 220 nM (lane 4) and were calculated as heterodimers.

Figure 3

Homologous-pairing activity of Xrcc3⋅Rad51C. A 32P-labeled single-stranded 120-mer oligonucleotide (1.6 μM) was incubated with Xrcc3⋅Rad51C in the standard reaction mixture at 37°C for 5 min, and the reactions were initiated by the addition of 12 mM MgCl2 and pGsat4 form I DNA (13 μM). (A) Time course experiments. The concentrations of Xrcc3⋅Rad51C (as heterodimers) and HsRad51 were 0.5 μM. The reaction times were 0 min (lanes 1 and 6), 5 min (lanes 2 and 7), 10 min (lanes 3 and 8), 20 min (lanes 4 and 9), and 40 min (lanes 5 and 10). (B) Graphic representation of time course experiments. The reactions were conducted with a 6.7-fold excess amount of ssDNA (molecule); the percentage of D-loop formation was calculated relative to the limiting amount of dsDNA. ●, Experiments with 0.5 μM Xrcc3⋅Rad51C; ○, control experiment without Xrcc3⋅Rad51C. (C) Graphic representation of protein titration experiments. The reactions were continued for 40 min. The reactions were conducted with a 6.7-fold excess amount of ssDNA (molecule); the percentage of D-loop formation was calculated relative to the limiting amount of dsDNA. ●, Experiments with homologous ssDNA and dsDNA; ○, control experiment with heterologous ssDNA and dsDNA. (D) Dissociation of D-loops by spontaneous branch migration. After a 40-min incubation with 0.5 μM Xrcc3⋅Rad51C, the D-loops formed by the reactions were treated with NheI (5 units) for 30 min at 37°C (lane 4) or pancreatic DNase (15 ng/ml) for 30 s at room temperature (lane 5). Lane 1 is a negative control without protein, and lane 2 is a control without DNase treatment. Lane 3 is a D-loop formed by the nonenzymatic method (36). (E) Homologous-pairing activity of Xrcc3⋅Rad51C between double-stranded and single-stranded oligonucleotides. A 32P-labeled single-stranded 50-mer oligonucleotide (600 nM) and a double-stranded 50-mer oligonucleotide (1.2 μM) were used as substrates. The concentration of Xrcc3⋅Rad51C was 200 nM. Reactions were continued for 0 min (lane 1), 10 min (lane 2), 30 min (lane 3), and 60 min (lane 4). Products were deproteinized and were analyzed by 12% PAGE.

Figure 4

Mg2+ and ATP requirements for homologous pairing by Xrcc3⋅Rad51C. (A) Mg2+ dependence of homologous pairing by Xrcc3⋅Rad51C. A 32P-labeled single-stranded 120-mer oligonucleotide (1.6 μM) was incubated with Xrcc3⋅Rad51C (0.5 μM) at 37°C for 5 min in the presence of various amounts of MgCl2. The reactions were initiated by the addition of pGsat4 form I DNA (13 μM) and were continued for 10 min. MgCl2 concentrations are 0 mM (lane 1), 1.25 mM (lane 2), 6.25 mM (lane 3), 13 mM (lane 4), 26 mM (lane 5), and 50 mM (lane 6). (B) ATP dependence of homologous pairing by Xrcc3⋅Rad51C. A 32P-labeled single-stranded 120-mer oligonucleotide (1.6 μM) was incubated with RecA (0.1 μM) or Xrcc3⋅Rad51C (0.5 μM) at 37°C for 5 min in the presence or absence of ATP, and the reactions were initiated by the addition of 12 mM MgCl2 and pGsat4 form I DNA (13 μM). Lanes 1–3 are reactions with ATP, and lanes 4–6 are reactions without ATP. Lanes 1 and 4 are negative controls without protein. Lanes 2 and 5 are the reactions with RecA, and lanes 3 and 6 are the reactions with Xrcc3⋅Rad51C.

Figure 5

DNA binding and electron microscopic visualizations of Xrcc3⋅Rad51C and Rad51C. (A) Superhelical dsDNA binding by Xrcc3⋅Rad51C and Rad51C. Superhelical dsDNA (10 μM) was incubated with Xrcc3⋅Rad51C or Rad51C at 37°C for 10 min, and the reactions were analyzed by 0.8% agarose gel electrophoresis in 0.5× TBE buffer. The concentrations of Xrcc3⋅Rad51C and Rad51C used in the DNA-binding experiments were 0.4 μM, 0.7 μM, and 1.0 μM. The concentration of Xrcc3⋅Rad51C was calculated as heterodimers. (B) Circular ssDNA binding by Xrcc3⋅Rad51C and Rad51C. Superhelical dsDNA (30 μM) was incubated with Xrcc3⋅Rad51C at 37°C for 10 min, and the reactions were analyzed by 0.8% agarose gel electrophoresis in 0.5× TBE buffer. The concentrations of Xrcc3⋅Rad51C and Rad51C used in the DNA-binding experiments were 0.4 μM, 0.7 μM, and 1.0 μM. The concentration of Xrcc3⋅Rad51C was calculated as heterodimers. (C) Electron microscopic visualization of Xrcc3⋅Rad51C complexed with ssDNA. The complexes were visualized by negative staining with uranyl acetate. (The magnification bar represents 100 nm.) (D) Electron microscopic visualization of Rad51C complexed with ssDNA. (E) Electron microscopic visualization of RecA complexed with ssDNA. (F) Electron microscopic visualization of HsRad51 complexed with ssDNA and dsDNA. (Left) HsRad51 complexed with ssDNA. (Right) HsRad51 complexed with dsDNA.

Figure 6

Homologous-pairing activity of Rad51C. A 32P-labeled single-stranded 50-mer oligonucleotide (1 μM) was incubated with Xrcc3⋅Rad51C in the standard reaction mixture at 37°C for 5 min, and the reactions were initiated by the addition of 12 mM MgCl2 and pGsat4 form I DNA (13 μM). The reactions were continued for 20 min. Lane 1 is the negative control without protein. Lanes 2–4 are the experiments with Xrcc3⋅Rad51C, and lanes 5–7 are the experiments with Rad51C alone. The concentrations of Xrcc3⋅Rad51C and Rad51C used in the DNA-binding experiments were 0.2 μM (lanes 2 and 5) and 0.6 μM (lanes 3, 4, 6, and 7). Lanes 4 and 7 are the negative controls with heterologous ssDNA and dsDNA. The concentration of Xrcc3⋅Rad51C was calculated as heterodimers.