WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks

Abstract

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link-induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, gamma H2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link-induced collapsed replication forks.

Figure 1.

Role of WRN in S-phase checkpoint. Control and WRN shRNA U-2 OS cells were treated with (A) PUVA (0.1 μg 8-methoxypsoralen/ml), (B) CPT (1 μM, 3 h), (C) HU (0.5 mM, 24 h), (D) ionizing radiation (6 Gy), (E) aphidicolin (1 μg/ml, 24 h), and (F) angelicin (0.1 μg/ml)+UVA. Cells were harvested at the indicated time points and analyzed by flow cytometry (n = 3–4).

Figure 2.

Quantification of cell cycle profiles as described in Figure 1. (A) PUVA; (B) CPT; (C) HU.

Figure 3.

Pulse-field gel electrophoresis of control shRNA U-2 OS cells. DNA from U-2 OS cells was analyzed by PFGE after exposure to DNA damaging agents as follows: 30 min after 6 Gy γ-irradiation, 24 h after PUVA treatment (0.1 μg 8-methoxypsoralen/ml), 16-h recovery after CPT treatment (1 μM for 3 h), 8-h recovery after HU treatment (0.5 mM for 24 h), and 6-h recovery after Aph treatment (1 μg/ml for 24 h).

Figure 4.

WRN-dependent ATM activation. (A) ATM autophosphorylation on Ser-1981 was quantified in control and WRN shRNA cells treated with PUVA (0.1 μg 8-methoxypsoralen/ml), HU (0.5 mM), or γ-irradiation. Cell lysates were prepared at the indicated time points as described for Western analyses (see Materials and Methods). (B) As in A, except Western blot was probed with antibodies to ATM substrates. (C) Phosphorylation of ATM substrates in control and WRN shRNA U-2 OS cells 16 h after treatment with PUVA (0.1 μg 8-methoxypsoralen/ml). Representative images were shown. (D) Fluorescence intensity after staining with the anti-phospho-(SQ/TQ) antibody was averaged over fields of cells using the softwares Axio Vision 3.1 and AIM Rel 3.2 (Zeiss).

Figure 5.

Role of WRN and ATM in intra-S-phase checkpoint in cells treated with PUVA and γ-irradiation. (A) Rate of DNA synthesis in control, WRN and ATM shRNA U-2 OS cells after exposure to PUVA (0.1 μg 8-methoxypsoralen/ml). Cells were harvested at the indicated time points (n = 3). DNA synthesis was measured as described in Materials and Methods. (B) Rate of DNA synthesis in control, WRN and ATM shRNA U-2 OS cells 2 h after exposure to the indicated doses of ionizing radiation (n = 3).

Figure 6.

Role of WRN in γH2AX formation in cells treated with PUVA and γ-irradiation. Control and WRN shRNA U-2 OS cells were treated with PUVA (0.1 μg 8-methoxypsoralen/ml), and ionizing radiation. (A) Cells were fixed and stained with mouse antibodies against γH2AX, followed by detection using confocal microscope. (B) Cell lysates were prepared at the indicated time points and were analyzed by Western analyses using antibodies against γH2AX and H2AX (see Materials and Methods).

Figure 7.

Convergence between WRN and ATM in the cellular response to PUVA. (A) Control and WRN shRNA U-2 OS cells were treated with PUVA (0.1 μg 8-methoxypsoralen/ml), ATM inhibitor (ATMi, KU55933, 10 μM), or both and harvested at 24 h. (B) Control and WRN shRNA U-2 OS cells were cotreated with nocodazole, PUVA, and ATMi as described in A.

Figure 8.

Model of the Role of WRN and ATM in response to collapsed replication forks or DSBs.