Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation

Abstract

TopBP1 and Claspin are adaptor proteins that facilitate phosphorylation of Chk1 by the ATR kinase in response to genotoxic stress. Despite their established requirement for Chk1 activation, the exact way in which TopBP1 and Claspin control Chk1 phosphorylation remains unclear. We show that TopBP1 tightly colocalizes with ATR in distinct nuclear subcompartments generated by DNA damage. Although depletion of TopBP1 by RNA interference (RNAi) strongly impaired phosphorylation of multiple ATR targets, including Chk1, Nbs1, Smc1, and H2AX, it did not interfere with ATR assembly at the sites of DNA damage. These findings challenge the current concept of ATR activation by recruitment to damaged DNA. In contrast, Claspin, like Chk1, remained distributed throughout the nucleus both before and after DNA damage. Consistently, the RNAi-mediated ablation of Claspin selectively abrogated ATR's ability to phosphorylate Chk1 but not other ATR targets. In addition, downregulation of Claspin mimicked Chk1 inactivation by inducing spontaneous DNA damage. Finally, we show that TopBP1 is required for the DNA damage-induced interaction between Claspin and Chk1. Together, these results suggest that while TopBP1 is a general regulator of ATR, Claspin operates downstream of TopBP1 to selectively regulate the Chk1-controlled branch of the genotoxic stress response.

FIG. 1.

Impact of TopBP1 and Claspin on ATR- and ATM-mediated phosphorylations. (A) Cells conditionally expressing shRNA to TopBP1 or Claspin were incubated (+) or not incubated (−) with doxycycline (Dox) and treated or not treated with 2 Gy IR, 10 J/m² of UV, or 2 mM HU. One hour after these treatments, cells were harvested and lysed. Total and phosphorylated (P) proteins were analyzed by immunoblotting with the indicated antibodies. (B) shTopBP1 and shClaspin cells were induced as described for panel A and treated or not treated with 10 J/m² of UV. Lysates were prepared 1 h later, and the efficiency of knockdown as well as the extent of H2AX phosphorylation was assayed by immunoblotting. (C) U-2-OS cells were transfected for 48 h with siRNAs targeting TopBP1 and Claspin, either individually or in combination, as indicated. The remaining dishes were transfected with control siRNA. One hour after exposure to 10 J/m² of UV, cells were lysed and the efficiency of knockdown and the extent of Chk1 phosphorylation were assayed by immunoblotting.

FIG. 2.

Redistribution of ATR, TopBP1, and Claspin in response to laser-generated DNA damage. (A) U-2-OS cells were grown on glass coverslips and incubated in the presence of 10 μM BrdU for 24 h. One hour after exposure to UV-A laser (see Materials and Methods), cells were fixed and immunostained for endogenous Claspin and γ-H2AX. In addition, the nuclear DNA was counterstained with ToPro3. (B) U-2-OS cells stably expressing GFP-ATR were treated as described for panel A and immunostained for endogenous TopBP1 and γ-H2AX. (C) shTopBP1 cells were incubated (+) or not incubated (−) for 72 h with doxycycline (Dox), treated as described for panel A, and immunostained with antibodies towards RPA and 53BP1 (the latter protein was used as a marker of DNA damage). (D) U-2-OS cells stably expressing GFP-ATR were transfected with control siRNA for 72 h, siRNA targeting TopBP1 for 72 h, or siRNA targeting Claspin for 24 h. BrdU was added to the cultures for the last 24 h. A representative field of cells from each culture was microirradiated as described for panel A, and the ability of GFP-ATR to accumulate at sites of DNA damage was assayed by confocal microscopy. Bar = 10 μm.

FIG. 3.

Downregulation of Claspin, but not that of TopBP1, triggers H2AX phosphorylation in replicating cells. (A) Cells conditionally expressing Claspin-targeting shRNA were grown on glass coverslips in the presence (+) or absence (−) of doxycycline (Dox) for 48 h. Subsequently, BrdU (25 μM) was added to the medium for 1 h to label S-phase cells. After fixation, the coverslips were treated with DNase to expose the incorporated BrdU and stained with antibodies towards Claspin, BrdU, and γ-H2AX. (B) shTopBP1 cells were treated as described for panel A, except that they were treated with doxycycline for 72 h. Bar = 10 μm.

FIG. 4.

DNA damage-induced interaction between Claspin and Chk1 depends upon TopBP1. (A) U-2-OS cells were transiently transfected with FLAG-Chk1 expression plasmid, incubated in the presence or absence of 10 mM caffeine (Caff.) for 1 h, and exposed (+) or not exposed (−) to 25 J/m² of UV. Cells were lysed after 1 h and immunoprecipitated (IP) with monoclonal FLAG antibody. The levels of Chk1 and Claspin in the immunoprecipitates were assayed by immunoblotting. (B) U-2-OS cells were transiently transfected with wild-type (WT), kinase-dead (KD), or S317A/S345A (2A) versions of FLAG-tagged Chk1, as indicated, and exposed to 25 J/m² of UV. FLAG immunoprecipitates were immunoblotted for Claspin and Chk1. (C) shTopBP1 cells were incubated or not incubated with doxycycline (Dox) for 72 h and transfected with wild-type FLAG-Chk1 construct. FLAG immunoprecipitates and lysates used for immunoprecipitation were immunoblotted for the indicated proteins.

FIG. 5.

Roles of TopBP1 and Claspin in the ATR-mediated DNA damage response. TopBP1 directly assists ATR in the phosphorylation of numerous downstream targets at sites of DNA damage, including Chk1, Nbs1, Smc1, and H2AX. Claspin, on the other hand, adds another layer of control in channeling ATR signaling to Chk1. Additionally, TopBP1 regulates Chk1 not only by directly stimulating its phosphorylation by ATR but also by facilitating activation of Claspin and potentiating its ability to bind Chk1.