Okano S et al. (2005), Translocation of XRCC1 and DNA ligase IIIalpha ...

XB-ART-53448

Nucleic Acids Res 2005 Jan 12;331:422-9. doi: 10.1093/nar/gki190.

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Translocation of XRCC1 and DNA ligase IIIalpha from centrosomes to chromosomes in response to DNA damage in mitotic human cells.

Okano S , Lan L , Tomkinson AE , Yasui A .

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DNA single-strand breaks (SSBs) are the most frequent lesions caused by oxidative DNA damage. They disrupt DNA replication, give rise to double-strand breaks and lead to cell death and genomic instability. It has been shown that the XRCC1 protein plays a key role in SSBs repair. We have recently shown in living human cells that XRCC1 accumulates at SSBs in a fully poly(ADP-ribose) (PAR) synthesis-dependent manner and that the accumulation of XRCC1 at SSBs is essential for further repair processes. Here, we show that XRCC1 and its partner protein, DNA ligase IIIalpha, localize at the centrosomes and their vicinity in metaphase cells and disappear during anaphase. Although the function of these proteins in centrosomes during metaphase is unknown, this centrosomal localization is PAR-dependent, because neither of the proteins is observed in the centrosomes in the presence of PAR polymerase inhibitors. On treatment of metaphase cells with H2O2, XRCC1 and DNA ligase IIIalpha translocate immediately from the centrosomes to mitotic chromosomes. These results show for the first time that the repair of SSBs is present in the early mitotic chromosomes and that there is a dynamic response of XRCC1 and DNA ligase IIIalpha to SSBs, in which these proteins are recruited from the centrosomes, where metaphase-dependent activation of PAR polymerase occurs, to mitotic chromosomes, by SSBs-dependent activation of PAR polymerase.

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Species referenced: Xenopus
Genes referenced: parp1 xpa xrcc1

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	Figure 1. Accumulation of XRCC1 and LIGIIIÎ± at SSBs after local UV-irradiation in XPA-UVDE cells. Co-localization of XRCC1 with LIGIIIÎ± was identified by double immunolabeling. Two minutes after local UV-irradiation (20 J/m2) cells were fixed with paraformaldehyde and co-stained with anti-XRCC1 antibody (red, upper row) and anti-LIGIIIÎ± antibody (green, middle row); the column c is for XPA-UVDE cells treated with DIQ, an inhibitor of PARP, before UV-irradiation. Co-localization of XRCC1 with LIGIIIÎ± appears yellow in overlay (bottom row).
	Figure 2. In situ visualization of GFP-DNA LIGIIIÎ±, GFP-DNA LIGIIIÎ² and XRCC1 before and after local UV-irradiation. GFPâDNA Ligase IIIÎ± (GFP-LIGIIIÎ±) and GFPâDNA Ligase IIIÎ² (GFP-LIGIIIÎ²) were transiently expressed in XPA-UVDE cells before UV-irradiation (20 J/m2). The fluorescence images of the cells stained with anti-XRCC1 (red, second upper row) and the images of GFP (green, uppermost row) are shown. Co-localization appears yellow (third row). GFP-tagged proteins were visualized 2 min after local UV-irradiation (20 J/m2). Column c is for DIQ treated cells. The fluorescent images of GFP were superimposed onto the Nomarsky images in the bottom row.
	Figure 3. XRCC1 in centrosomes of HeLa cells during interphase. (A) Fluorescent micrographs of HeLa cells in interphase obtained by double immunolabeling for Î³-tubulin and XRCC1. Cells were fixed with methanol:acetone and co-stained with anti-XRCC1 antibody (a), and anti-Î³-tubulin antibody (b). XRCC1 and Î³-tubulin appear red and green, respectively. The corresponding Nomarsky image is shown in (c). Co-localization of both XRCC1 and Î³-tubulin appears yellow in overlay (d). (B) Fluorescent micrographs of HeLa cells in interphase expressing XRCC1-GFP. The cells were fixed with paraformaldehyde. DNA was stained with TOPRO-3 (middle panel) and superimposed onto the fluorescent image in the uppermost panel. Arrows indicate the position of centrosomes.
	Figure 4. Co-localization of XRCC1 in the centrosomes of HeLa cells from different mitotic phases. (A) Double immunolabeling for XRCC1 and Î³-tubulin. The cells were fixed with methanol:acetone and stained with anti-XRCC1 (red) and anti-Î³-tubulin (green) antibodies. Co-localization of the proteins appears yellow in the third row after overlay. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row). (B) Double immunolabeling for LIGIIIÎ± and XRCC1. The cells were fixed with paraformaldehyde and stained with anti-XRCC1 (red) and anti-LIGIIIÎ± (green) anti-bodies. Co-localization of the proteins appears yellow. (C) Double immunolabeling for PAR and XRCC1. The cells were fixed with methanol:acetone, and co-stained with anti-XRCC1 (red) and anti-PAR (green) antibodies. Panels in the first, the second and the third column are for untreated cells in prometapahase, metapahase and anaphase, respectively. Co-localization of the proteins appears yellow. DNA was stained with TOPRO-3 and superimposed onto the fluorescent images of the uppermost row (the bottom row).
	Figure 5. Distribution of XRCC1 in human cell lines. The upper left panel: fluorescent micrograph of HeLa cell in prophase by immunolabeling for XRCC1. The cells were fixed with paraformaldehyde, and stained with anti-XRCC1 monoclonal antibody. The lower left panel: fluorescent micrograph of AT5BI-VA cell in metaphase by immunolabeling for XRCC1. The cells were fixed with methanol:acetone, and stained with anti-XRCC1 antibody. The DNA was stained with TOPRO-3 and the corresponding images were superimposed onto the fluorescent images in the left columns and shown in the right columns.

References [+] :

Augustin, PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression. 2003, Pubmed