Fig. 5: DONSON stabilizes the CMG helicase during S phase to maintain the intrinsic S/G2 checkpoint.

a Protein expression levels of ETAA1 in G1, S, G2, and M phases of WT cells. HeLa cells were synchronized in G2 phase with 10 µM RO-3306 (Cdk1i), released, and harvested at the indicated time points. b A schematic of the DNA replication termination based on previous studies. c Representative immunoblot analysis of whole cell extracts for ETAA1 from HeLa cells treated with 20 μM P22077 (USP7i) or 10 µM NMS-873 (p97i) for 6 h. α-Tubulin was used as a loading control. d Representative immunoblot analysis of whole cell extracts for ETAA1 from HeLa cells treated with siControl or siDONSON for 48 h, followed by treatment with DMSO or 10 µM NMS-873 (p97i) for 6 h. e The amount of chromatin-bound Cdc45 in S phase upon DONSON depletion. HeLa cells were treated with siControl or siDONSON for 48 h, and soluble proteins were pre-extracted by PBS containing 0.2% Triton X-100 on ice for 10 s before fixation. f Quantification of the normalized signal intensity of chromatin-bound Cdc45 in S phase observed in (e). n = 50 from three independent experiments. g The amount of chromatin-bound Cdc45 in S phase upon DONSON depletion. HeLa cells were treated with siControl or siDONSON for 48 h and synchronized at the G1/S boundary with 6 µM Aphidicolin for 24 h, released and harvested at the indicated time points. Soluble proteins were pre-extracted by PBS containing 0.2% Triton X-100 on ice for 10 s before fixation. h Quantification of the normalized signal intensity of Cdc45 in the chromatin observed in (g). n = 50 from three independent experiments. i The amount of chromatin-bound p97 in S phase upon DONSON depletion. HeLa cells were treated with siControl or siDONSON for 48 h, and soluble proteins were pre-extracted by CSK buffer on ice for 20 s before fixation. j Quantification of the normalized signal intensity of chromatin-bound p97 in S phase observed in (i). n = 50 from three independent experiments. k The amount of chromatin-bound Cdc45 in S phase upon DONSON depletion. HeLa cells were treated with siDONSON for 48 h, and with or without NMS-873 for 6 h, and soluble proteins were pre-extracted by PBS containing 0.2% Triton X-100 on ice for 10 s before fixation. l Quantification of the normalized signal intensity of Cdc45 in the chromatin in S phase observed in (k). n = 50 from three independent experiments. m Phosphorylation of Plk1 at T210 residue in S phase. HeLa cells were treated with siDONSON for 48 h, and with or without NMS-873 for 6 h before fixation. n Quantification of the normalized signal intensity of phosphorylated Plk1 (T210) at each centriole observed in (m). n = 50 from three independent experiments. o Schematic model of the phenotypes of DONSON depletion. During DNA replication, ETAA1 localized to chromatin through its interaction with the single-stranded DNA-binding protein RPA⁶⁷. Depletion of DONSON causes precocious CMG disassembly and following ETAA1 degradation in S phase. All scale bars, 5 μm. Values are mean percentages ± s.d. Mann–Whitney U test was used in (f), (j), (l), and (n), and the Kruskal–Wallis test was used in (h) to obtain the P-value. Source data are provided as a Source Data file.