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. 2006 Jun 7;25(11):2605-14.
doi: 10.1038/sj.emboj.7601123. Epub 2006 May 4.

Opposing effects of the UV lesion repair protein XPA and UV bypass polymerase eta on ATR checkpoint signaling

Ryan D Bomgarden  1 , Patrick J Lupardus, Deena V Soni, Muh-Ching Yee, James M Ford, Karlene A Cimprich
Affiliations

Opposing effects of the UV lesion repair protein XPA and UV bypass polymerase eta on ATR checkpoint signaling

Ryan D Bomgarden et al. EMBO J. .

Erratum in

  • EMBO J. 2006 Oct 18;25(20):5036

Abstract

An essential component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV-induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation.

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Figures

Figure 1
Figure 1
XPA facilitates ATRIP recruitment to sites of UV damage. HeLa cells grown on coverslips were exposed to 50 J/m2 UV through a porous membrane and allowed to recover for 1 h. Cells were fixed and stained with an anti-ATRIP rabbit polyclonal antibody and an anti-CPD mouse monoclonal antibody (A) or an anti-XPA mouse monoclonal antibody (B), followed by staining with a fluorescent goat anti-rabbit antibody Alexa 488 (green) and goat anti-mouse antibody Alexa 595 (red). Nuclei were counterstained with DAPI. Colocalization of labeled proteins is shown as yellow in merge. (C) XPA-complemented cells (gray bar) and XPA-deficient cells (white bar) were UV treated and stained as in panel (A) at the times shown. The recruitment of ATRIP to CPD lesions was determined for a total of 200 nuclei containing CPD foci. Shown graphically is the percentage of nuclei with ATRIP foci that colocalized with CPD foci. Error bars indicate standard error of three samples from a representative experiment (n=3) performed in triplicate. (D) Percentage of S-phase cells was determined by flow cytometry using propidium iodide staining (top panels) and by measuring BrdU incorporation (bottom panels).
Figure 2
Figure 2
XPA-deficient cells show reduced Chk1 phosphorylation following UV treatment. (A) Isogenic XPA-deficient and XPA-complemented cells were either mock treated or exposed to 0, 5, 15 or 50 J/m2 UV. Cells were harvested after 1 h and lysates were resolved by SDS–PAGE and analyzed by Western blot using phospho-serine 345 Chk1, Chk1 and ATRIP antibodies. (B) XPA-complemented and XPA-deficient cells were mock treated or exposed to 15 J/m2 UV and then harvested at the times shown. Lysates were analyzed as described in panel A. (C) HeLa cells were transfected with control siRNA or one of two siRNAs to XPA and treated with 15 J/m2 UV 48 h later. At 1 h after UV treatment, lysates were prepared and resolved by SDS–PAGE and analyzed by Western blot using phospho-serine 345 Chk1, Chk1, ATRIP and XPA antibodies.
Figure 3
Figure 3
XPA is required for full Chk1 phosphorylation induced by 4-NQO, but not aphidicolin or IR. Isogenic XPA-complemented and XPA-deficient cells were exposed to increasing concentrations of the UV-mimetic drug 4-NQO for 1 h (A), 5 μM aphidicolin for 6 h (B) or 10 Gy IR followed by a 2 h recovery (C). Lysates were prepared and analyzed as described in Figure 2.
Figure 4
Figure 4
Depletion of XPA from Xenopus egg extracts inhibits UV-induced Chk1 phosphorylation. (A) Xenopus egg extracts were incubated with either UV-damaged sperm chromatin (1000 J/m2) or treated with 150 μM aphidicolin. After 90 min, total extract samples were taken and an aliquot was removed for analysis of Chk1 S344 phosphorylation. From the remaining sample, chromatin was isolated through a sucrose cushion, sheared and boiled in sample buffer. Samples were resolved by SDS–PAGE and analyzed by Western blot using the indicated antibodies. (B) Xenopus extract was either mock-depleted or XPA-depleted using rabbit IgG (mock) or an xXPA antibody coupled to protein A beads. Undamaged or UV-treated sperm chromatin was added to the extracts and incubated for 90 min before addition of sample buffer. Equal amounts of extract were resolved by SDS–PAGE and analyzed by Western blot using the indicated antibodies.
Figure 5
Figure 5
ATRIP translocation and Chk1 phosphorylation are unaffected by loss of XPC or CSB. Isogenic XPC-deficient (white bar) and XPC-complemented (gray bar) cells (A) or CSB-deficient (white bar) and CSB-complemented (gray bar) cells (C) were UV treated and stained as in Figure 1A. Colocalization of ATRIP and CPD was determined as in Figure 1C. Percentage of S-phase cells was determined by flow cytometry as shown in Figure 1D. XPC-deficient and XPC-complemented cells (B) or CSB-deficient and CSB-complemented cells (D) were treated with increasing amounts of UV and allowed to recover for 1 h. Equal amounts of cell lysate were resolved by SDS–PAGE and analyzed by Western blot using the indicated antibodies.
Figure 6
Figure 6
XPF and XPG are not required for UV-induced Chk1 phosphorylation. Isogenic XPF-deficient and XPF-complemented cells (A) or XPG-deficient and XPG-complemented cells (B) were either mock treated or exposed to UV radiation and allowed to recover for 1 h. Equal amounts of cell lysate were resolved by SDS–PAGE and analyzed by Western blot using the indicated antibodies.
Figure 7
Figure 7
RPA chromatin binding and phosphorylation is decreased in XPA-deficient cells. XPA-deficient and XPA-complemented cells were treated with increasing amounts of UV radiation and fractionated. Proteins of the insoluble chromatin fraction were resolved by SDS–PAGE and analyzed by Western blot using the indicated antibodies.
Figure 8
Figure 8
Chk1 and RPA phosphorylation are increased in XPV cells. (A) Polη-deficient (XP-V) and Polη-complemented (XP-V+Polη) cells were either mock treated or exposed to 15 J/m2 UV and allowed to recover for the times indicated. Equal amounts of cell lysate were resolved by SDS–PAGE and analyzed by Western blot using the indicated antibodies. (B) Polη-deficient and Polη-complemented cells were treated with 5 μM aphidicolin for 6 h and then analyzed as described in panel A. (C) Polη-deficient and Polη-complemented cells were treated with increasing amounts of UV and fractionated. Proteins of the insoluble chromatin fraction were resolved by SDS–PAGE and analyzed by Western blot using the indicated antibodies.
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