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First published online 27 February 2007
doi: 10.1242/jcs.03391

Journal of Cell Science 120, 1104-1112 (2007)
Published by The Company of Biologists 2007
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Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells

Megumi Matsumoto1, Kie Yaginuma1, Ai Igarashi1, Mayumi Imura1, Mizuho Hasegawa1, Kuniyoshi Iwabuchi2, Takayasu Date2, Toshio Mori3, Kanji Ishizaki4, Katsumi Yamashita1, Manabu Inobe1 and Tsukasa Matsunaga1,*

1 Laboratory of Human Molecular Genetics, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan
2 Department of Biochemistry, Kanazawa Medical University, Ishikawa 920-0293, Japan
3 Radioisotope Research Center, Nara Medical University, Kashihara 634-8521, Japan
4 Central Laboratory and Radiation Biology, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan


Figure 1
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  		Fig. 1. UV-induced H2AX phosphorylation occurs independently in quiescent cells and cycling S-phase cells. (A) MSU-2 cells growing asynchronously (AS) or growth-arrested by contact inhibition and serum starvation (G0) were irradiated with 10 J/m2 UV, incubated for 4 hours and treated with 50 µM BrdU for 15 minutes before fixation. Cells were stained with anti-{gamma}-H2AX and anti-BrdU antibodies and DAPI was used for nuclear counterstaining. Note that the BrdU-labeling index was less than 1% in the growth-arrested population. (B) MSU-2 cells (AS or G0) were irradiated with UV (black line, 0 J/m2; red line, 10 J/m2), incubated for 1 hour and treated with 50 µM BrdU for 30 minutes before fixation. Cells were stained with anti-{gamma}-H2AX and anti-BrdU antibodies, and PI staining was performed to measure cellular DNA content. For the asynchronous population, cells were divided into three subpopulations (G1, 65%; S, 19%; G2/M, 14%) based on the staining with PI and anti-BrdU antibody, and histograms of {gamma}-H2AX intensity in each subpopulation are shown in the upper panels. For quiescent cells, a BrdU-negative 2N subpopulation (92%) was analyzed and shown as a histogram of {gamma}-H2AX in the lower panel. (C) Growth-arrested MSU-2 cells were released by subculturing at lower density under normal serum conditions and irradiated with 10 J/m2 UV at 12, 15 or 18 hours after the release. The cells were incubated for 30 minutes and processed for immunostaining as described in A.

 

Figure 2
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  		Fig. 2. UV-induced H2AX phosphorylation in quiescent cells occurs at damaged DNA sites and correlates with repair kinetics of UV lesions. (A,B) Growth-arrested MSU-2 cells were locally irradiated with 100 J/m2 UV through an isopore membrane filter and incubated for various periods. Cells were fixed and stained with anti-{gamma}-H2AX and anti-CPD antibodies (A), or anti-6-4PP antibody alone (B). (C) MSU-2 cells growing asynchronously (closed symbols) or growth-arrested (open symbols) were irradiated with 10 J/m2 UV and incubated for the indicated periods. Genomic DNA was isolated and the amounts of CPD and 6-4PP were determined using an enzyme-linked immunosorbent assay. Each point represents the mean of three experiments and bars indicate the s.d.

 

Figure 3
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  		Fig. 3. Nucleotide excision repair is required for the UV-induced H2AX phosphorylation in quiescent cells. (A) MSU-2 or XP2BI cells (AS or G0) were irradiated with 10 J/m2 UV and incubated for 4 hours. 50 µM BrdU was included in the asynchronous population for 15 minutes before fixation and cells were costained with anti-{gamma}-H2AX and anti-BrdU antibodies. (B) MSU-2 or XP2BI cells under growth-arrested conditions were locally irradiated with 40 J/m2 UV and incubated for 4 hours. Cells were fixed and double-stained with anti-{gamma}-H2AX and anti-CPD antibodies. (C) MSU-2, TIG-120, XP12BE, XP1CTA and XP2BI cells growth-arrested by contact inhibition and serum starvation were irradiated with no UV (black lines) or 10 J/m2 UV (red lines) and incubated for 1 hour before fixation. Cells were stained with anti-{gamma}-H2AX and PI and analyzed by flow cytometry. (D) XP3OS/T-n cells were transfected with pCMV-Myc-XPA plasmid using TransFectin lipid reagent (Bio-Rad) and incubated for 2 days. The cells were growth-arrested by serum starvation for 4 days and exposed to 20 J/m2 UV. After 3 hours of incubation, cells were fixed and double-stained with anit-{gamma}-H2AX and anti-6-4PP antibodies. (E) MSU-2 or XP2BI cells (AS or G0) were treated with 2 µM NA-AAF for 30 minutes and incubated for another 30 minutes after medium change. BrdU was used for labeling S-phase cells in the AS population, and {gamma}-H2AX and BrdU were detected as described in A.

 

Figure 4
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  		Fig. 4. Perturbation of repair replication in NER enhances H2AX phosphorylation. (A,B) MSU-2 or XP2BI cells under growtharrested conditions were exposed to 10 J/m2 UV globally (A) or locally (B), incubated for 1 hour in the absence or presence of 100 µM Ara-C before fixation and stained with anti-{gamma}-H2AX antibody. (C) Growth-arrested MSU-2 cells were exposed to 10 J/m2 UV or 15 µg/mL etoposide and incubated for 1 hour in the presence (red lines) or absence (black lines) of Ara-C before fixation. Cells were stained with anti-{gamma}-H2AX antibody and PI and analyzed by flow cytometry.

 

Figure 5
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  		Fig. 5. Cellular levels of repair replication factors are markedly reduced in quiescent cells. (A) MSU-2 or XP2BI cells (AS or G0) were lysed and cellular levels of the indicated proteins were analyzed by immunoblotting with specific antibodies. (B) Growth-arrested MSU-2 cells were released as described in the legend of Fig. 1C. Cell lysates were prepared at the indicated time points and used for immunoblotting with anti-DNA Pol {delta} (125-kDa CS) or anti-DNA Pol {epsilon} (261-kDa CS) antibodies. The quantitative data are shown in the bottom panel and represent the relative protein levels (%) to the AS sample.

 

Figure 6
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  		Fig. 6. ATR is the principal kinase for the NER-dependent H2AX phosphorylation. (A,B) MSU-2 or GM18366 (ATR-Seckel syndrome) cells under growth-arrested conditions were exposed to 10 J/m2 UV and incubated for 4 hours (A), or treated with 40 µg/mL etoposide for 1 hour (B), and stained with anti-{gamma}-H2AX antibody. (C,D) MSU-2 or XP2BI cells under growtharrested conditions were exposed to local UV (40 J/m2), and incubated for 1 hour in the presence or absence of 100 µM Ara-C before staining with anti-RPA2 (p34) antibody (C) or incubated for 1 hour with 100 µM Ara-C before doublestaining with anti-ATRIP and anti-CPD antibodies (D).

 

Figure 7
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  		Fig. 7. The NER-dependent H2AX phosphorylation coincides with 53BP1 accumulation at damaged sites but not with Chk1 Ser345 phosphorylation. (A) MSU-2 cells (AS or G0) were exposed to 20 J/m2 UV and incubated for 2 or 4 hours. Cell lysates were prepared with SDS sample buffer (Bio-Rad) and analyzed by immunoblotting with the specific antibodies as indicated. (B) MSU-2 or XP1CTA cells under growth-arrested conditions were locally irradiated with 20 J/m2 UV and stained with anti-53BP1 antibody after 4 hours of incubation. (C) Growth-arrested MSU-2 cells were locally irradiated with 10 J/m2 UV, incubated for 1 hour in the presence or absence of Ara-C and stained with anti-53BP1 antibody.

 

Figure 8
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  		Fig. 8. Working model for the NER-dependent H2AX phosphorylation in G0/G1-arrested human cells. Two distinct but partially overlapping pathways can be activated by UV or NA-AAF: one is a replication stress-induced pathway in cycling S-phase cells (Ward and Chen, 2001Go; Ward et al., 2004Go) and the other is a NER-mediated pathway in quiescent cells (this study). In both pathways ATR is the principal kinase for the H2AX phosphorylation and RPA seems to play a role in the recruitment of ATR-ATRIP. A major difference between the two pathways is in how RPA-coated ssDNA regions are generated. The NER-mediated ssDNA gaps or their processed products might be caused by perturbed repair synthesis because of extremely low levels of replication factors including Pol {delta}, Pol {epsilon} and PCNA (shown in grey).

 

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© The Company of Biologists Ltd 2007