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, 23 (23), 4660-9

Interaction Between Human MCM7 and Rad17 Proteins Is Required for Replication Checkpoint Signaling

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Interaction Between Human MCM7 and Rad17 Proteins Is Required for Replication Checkpoint Signaling

Cheng-Chung Tsao et al. EMBO J.

Abstract

Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.

Figures

Figure 1
Figure 1
Co-immunoprecipitation of hRad17 with hMCM7. (A) Co-immunoprecipitation of FLAG-tagged hRad17 and HA-tagged hMCM7. HeLa cells were cotransfected with the indicated plasmids, and cellular extracts (0.5 mg protein) were immunoprecipitated (IP) with α-FLAG or α-HA antibodies. (B) Genotoxic stress-induced co-immunoprecipitation of hRad17, hMCM7, and ATRIP. HeLa cells were exposed for 4 h to 200 J/m2 UV-B light or 1 μM Aph. Cellular extracts (1 mg protein) were immunoprecipitated and immunoblotted with the indicated antibodies.
Figure 2
Figure 2
Depletion of hMCM7 and hRad17 inhibits genotoxic stress-induced hChk1 phosphorylation. (A) HeLa cells were transfected with hMCM7 or hRad17 siRNA, and then treated with 200 J/m2 of UV or 20 Gy IR at 48 h post-transfection. Cellular extracts were harvested at 1 h after UV or IR treatment, and cell extracts (50 μg protein) were immunoblotted with the indicated antibodies. β-Tubulin served as a sample-loading control. (B) hMCM7, hMCM2, or hRad17 siRNA-transfected HeLa cells were treated for 4 h with 200 J/m2 UV light or 1 μM Aph, and cellular extracts were separated by SDS–PAGE and immunoblotted with the indicated antibodies. (C) Cells were transfected with hMCM7 or hMCM2 siRNA, and were treated with 1 μM Aph. Cells were fixed after 4 h and stained with the indicated antibodies. Cell nuclei were stained with DAPI.
Figure 3
Figure 3
Loss of hMCM7 interferes with the formation ATR nuclear foci in UV-damaged cells. (A) Immunofluorescence microscopy. HeLa cells were either left untreated or irradiated with 200 J/m2 UV light. Cells were fixed after 6 h, and immunostained with α-ATR or α-hMCM7 antibodies. In the bottom two rows, the cells were transfected with the indicated siRNAs, and were irradiated with UV light after 48 h. DAPI staining was used to mark cell nuclei. (B) Quantitation of ATR nuclear foci in the cell populations shown in panel A. Triplicate samples (minimum, 100 cells per sample) were counted manually by microscopy. Error bars indicate standard error of the mean from the three sample populations.
Figure 4
Figure 4
hMCM7 depletion confers a UVDS phenotype. (A) Time course of UVDS. A549 cells were treated with the indicated siRNAs, and, after 48 h, were irradiated with 200 J/m2 UV light. DNA synthesis was measured at the indicated times after UV light exposure as described in Materials and methods. Error bars indicate standard deviation from quadruplicate samples. (B) Effect of hMCM7 versus hRad17 siRNA treatment on UVDS. A549 cells were treated with the indicated siRNAs, and were irradiated with 200 J/m2 UV light at 48 h post-transfection. DNA synthesis was determined at 1 h after radiation exposure. (C) Effect of hMCM7 versus or hRad17 depletion on RDS. A549 cells were treated with the indicated siRNAs, and were irradiated with 20 Gy IR at 48 h post-transfection. DNA synthesis was determined at 1 h after radiation exposure.
Figure 5
Figure 5
Effects of hMCM7 or hRad17 depletion on UV-induced S-phase checkpoint activation and cell survival. (A) Impaired S-phase checkpoint in hMCM7-depleted cells. HeLa cells were transfected with the indicated siRNAs, and then exposed to 200 J/m2 UV light at 48 h after transfection. Cell-cycle distributions were examined at 24 h postirradiation. (B) Cell survival after UV irradiation. HeLa cells were transfected with indicated siRNA and, after 48 h, were exposed to the indicated doses of UV light. Cell survival was determined after 24 or 48 h in triplicate samples with an MTT dye conversion assay. Absorbance values were normalized to the zero time control for each cell population, which was arbitrarily set at 100%. Error bars indicate standard deviations. (C) Effect of hMCM7 or hRad17 depletion on cellular recovery from an Aph block. HeLa cells were transfected with the indicated siRNAs, and were replated after 24 h for survival assays. After 24 h in culture, the cells were treated for 16 h with 1 μM Aph, and then released into drug-free medium. Cell survival was determined in quadruplicate samples as described in Materials and methods. Values were normalized to the untreated control for each cell population, and error bars indicate standard deviations.
Figure 6
Figure 6
Disruption of S-phase checkpoint signaling by ectopic expression of the hMCM7-binding region of hRad17. (A) Binding of ectopically expressed GFP-Rad17-5 to endogenous hMCM7. HeLa cells were transfected with GFP or GFP-Rad17-5 fusion construct (encoding amino acids 421–670 of hRad17). At 24 h post-transfection, the cells were harvested, lysed, and detergent-soluble protein (1 mg) was immunoprecipitated with α-hMCM7 antibody. (B) hChk1 phosphorylation. HeLa cells were transfected with GFP or GFP-Rad17-5, and then treated with 200 J/m2 UV light or 1 μM Aph at 16 h after transfection. Cellular extracts (50 μg protein) were prepared after 2 h, and proteins were immunoblotted with the indicated antibodies. (C) Ectopically expressed GFP-Rad17-5 confers a UVDS phenotype. HeLa cells were transiently transfected with either GFP- or GFP-Rad17-5-encoding plasmids, and, after 24 h, were irradiated with 200 J/m2 UV light. DNA synthesis was determined at 1 h after radiation exposure as described in Materials and methods. (D) hChk2 phosphorylation. HeLa cells were transfected with GFP or GFP-Rad17-5, and then treated with 200 J/m2 UV light or 20 Gy of IR at 16 h after transfection. Cellular extracts (50 μg protein) were prepared after 2 h, and proteins were immunoblotted with the indicated antibodies.
Figure 7
Figure 7
Histone H2AX phosphorylation. (A) γH2AX staining. HeLa cells were transiently transfected with either GFP- or GFP-Rad17-5-encoding plasmids, then were fixed and permeabilized after 16 h in culture. The cells were stained with γH2AX-specific antibody (red), and cell nuclei were counterstained with DAPI (blue). (B) Quantitation of γH2AX-positive cell nuclei. A minimum of 100 cells from each of the cell populations depicted in panel A were scored for nuclear γH2AX staining. (C) γH2AX staining in hMCM7- versus hRad17-depleted HeLa cells. The indicated siRNA-transfected cells were costained with α-γH2AX antibody (red) and either hRad17 or hMCM7 antibody (green) after 60 h in culture. Cell nuclei were counterstained with DAPI (blue).

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