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. Author manuscript; available in PMC: 2021 Mar 9.
Published in final edited form as: Cell Rep. 2021 Jan 26;34(4):108680. doi: 10.1016/j.celrep.2020.108680

Figure 5. Chk1 promotes NHEJ by facilitating histone ubiquitination on H2AX and 53BP1 localization at DSBs in G1 phase and is itself activated by ATM.

Figure 5.

(A) Chk1 is required for NHEJ repair. NHEJ efficiency was measured as described in Figure 2G, and the decrease of NHEJ efficiency was calculated by subtracting the value in each siRNA or inhibitor treatment from controls (siGL2 or DMSO). 293B cells were transfected with siGL2 or indicated siRNA twice at 24-h intervals. 5 µM MK-8776 and 600 nM UCN-01 were added at 20 h and 5 h before harvest, respectively. Mean ± SD of triplicates.

(B and C) Chk1 inhibition decreases 53BP1 foci in G1 and S/G2 cells. U2OS cells were treated with 20 µM MK8776 for 1 h followed by 20 µg/mL bleomycin for additional 1 h. Representative images (B) and quantitation of 53BP1 foci-positive cells in cyclin A-negative (G1) and -negative (S/G2) cells (C) are shown. Cells in dashed lines indicate cyclin A-negative cells. Mean ± SD of triplicates. One-way ANOVA (Tukey’s post hoc test); **p < 0.01; ****p < 0.0001. Scale bar, 10 µm.

(D) Histone ubiquitination suppressed by Chk1 inhibition in G1, not S/G2 phase, on DSBs. U2OS cells were treated with 20 µM MK8776 for 1 h followed by 20 µg/mL bleomycin for additional 1 h in G1- or S/G2-enriched cells. Decreased autophosphorylation of Chk1 at S296 (pS296-Chk1), used as a measure of Chk1 activity.

(E) ATM-dependent phosphorylation of Chk1 in G1 phase on DSBs. HeLa DR13–9 cells were accumulated in G1 phase from nocodazole release followed by 20 µg/mL bleomycin for 1 h. Cell-cycle profiles by fluorescence-activated cell sorting (FACS) analysis at the bottom.

(F) ASF1A interaction with MDC1 requires Chk1 activity and the S166 phosphorylation in G1 phase. HEK293T cells were transfected by HA-ASF1A WT or S166A plasmids and accumulated in G1 by nocodazole release. 500 nM UCN-01 was treated for 1 h followed by bleomycin treatment. Decreased phosphorylation of TLK1 at S695 (pS695-TLK1) was used as a measure for Chk1 inactivation.

(G) Co-immunoprecipitation of ASF1A with Chk1 seen in G1 phase, but not in S phase.