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. 2002;2(2):106-113.
doi: 10.1155/S1110724302204040.

XRCC2 is Required for the Formation of Rad51 Foci Induced by Ionizing Radiation and DNA Cross-Linking Agent Mitomycin C

Nan Liu

XRCC2 is Required for the Formation of Rad51 Foci Induced by Ionizing Radiation and DNA Cross-Linking Agent Mitomycin C

Nan Liu. J Biomed Biotechnol. 2002.

Abstract

XRCC2 protein shares weak amino acid sequence similarity with Rad51, which is a central player in homologous recombinational repair (HRR). Rad51 proteins assemble at the sites of HRR and form visible nuclear foci in response to DNA damage. Xrcc2 hamster mutant irs1 cells are incapable of forming Rad51 foci after ionizing irradiation or DNA cross-linking agent mitomycin C treatment, though the Rad51 protein level is normal in the mutant. The defect can be corrected in an XRCC2 transformant. Time course study showed that the irs1 cells primarily lacked the early response (2 hours after irradiation) to form small Rad51 foci (type 1) and later response (8 hours after irradiation) to form large foci (type 2). These results suggested that XRCC2 is essential for the assembly of the DNA damage-induced Rad51 foci and that XRCC2 may play an important role in the early stage of HRR.

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Figures

Figure 1
Figure 1
Rad51 focus formation induced by γ-ray irradiation. (a) Immunostaining with mouse α-Rad51 antibody of wild-type V79 and xrcc2 mutant irs1 nuclei 2 hours after 8 Gy γ-irradiation. The nuclei containing Rad51 foci are indicated with arrows. (b) Percentage of cells containing Rad51 foci before and after γ-ray irradiation in HeLa, V79, irs1, XRCC2 cDNA transformant pDXR2, and genomic transformant GT621. At least 200 nuclei for each sample were scored in each experiment and a threshold of 5 foci/cell was used. Error bars indicate the standard deviations of the average values from two independent experiments. One experiment was done for pDXR2 and GT621.
Figure 1
Figure 1
Rad51 focus formation induced by γ-ray irradiation. (a) Immunostaining with mouse α-Rad51 antibody of wild-type V79 and xrcc2 mutant irs1 nuclei 2 hours after 8 Gy γ-irradiation. The nuclei containing Rad51 foci are indicated with arrows. (b) Percentage of cells containing Rad51 foci before and after γ-ray irradiation in HeLa, V79, irs1, XRCC2 cDNA transformant pDXR2, and genomic transformant GT621. At least 200 nuclei for each sample were scored in each experiment and a threshold of 5 foci/cell was used. Error bars indicate the standard deviations of the average values from two independent experiments. One experiment was done for pDXR2 and GT621.
Figure 2
Figure 2
Induction of Rad51 nuclear foci by MMC treatment. HeLa, V79, irs1, pDXR2, and GT621 cells were incubated with MMC at various concentrations at 37°C for 16 hours.
Figure 3
Figure 3
Western blot with α-MmRad51 antibody. Cell extracts (40 μg) of V79, irs1, or pDXR2 was loaded and proteins were seperated on 10% SDS-PAGE gel. The hamster Rad51 (37 kd) is indicated.
Figure 4
Figure 4
Time course of Rad51 focus formation. (a) Combined image of FITC (Rad51) and DAPI (nuclei) staining at 0.5, 4, 8, and 24 hours after irradiation. Irs1 (top panels) and V79 (bottom panels) cells were irradiated with 8 Gy γ-rays and incubated at 37°C for various times. The irs1 nuclei contain the small foci (type I foci) are indicated with arrows. (b) Percentage of cells containing Rad51 foci at the time points after irradiation.
Figure 4
Figure 4
Time course of Rad51 focus formation. (a) Combined image of FITC (Rad51) and DAPI (nuclei) staining at 0.5, 4, 8, and 24 hours after irradiation. Irs1 (top panels) and V79 (bottom panels) cells were irradiated with 8 Gy γ-rays and incubated at 37°C for various times. The irs1 nuclei contain the small foci (type I foci) are indicated with arrows. (b) Percentage of cells containing Rad51 foci at the time points after irradiation.
Figure 5
Figure 5
(a) Hamster nuclei containing type I and type II Rad51 foci (top panels). The correspondent nuclei stained with DAPI are shown in bottom. (b) Percentage of cells containing type I or type II foci in V79 (left) and irs1 (right) cells irradiated with 8 Gy γ-rays and incubated at 37°C for various times.
Figure 5
Figure 5
(a) Hamster nuclei containing type I and type II Rad51 foci (top panels). The correspondent nuclei stained with DAPI are shown in bottom. (b) Percentage of cells containing type I or type II foci in V79 (left) and irs1 (right) cells irradiated with 8 Gy γ-rays and incubated at 37°C for various times.
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