Fig. 6: The BAHCC1^TTD:H4K20me1 interaction is required for proper S-phase progression.

a, b Flow cytometric analysis of DNA synthesis by EdU pulse labeling and DNA content (a) and quantification of EdU incorporation in S phase (b) using HeLa cells with BAHCC1 KO (right) or its TTD-dead/Y1993A mutation (middle), relative to WT (left). Two-tailed unpaired t-test in (b): ****P < 0.0001; n = 4,800 (WT), 4,053 (Y1993A) and 3,805 (KO) S phase cells determined by flow cytometry. Values represent means ± SD. c, d Flow cytometric analysis of EdU labeling (c) and quantification of EdU labeling in S phase (d) using MEFs bearing WT Bahcc1 (left) or its TTD-dead mutant (right; Y1998A). Two-tailed unpaired t-test in (d): ****P < 0.0001; n = 2,549 (WT) and 3,166 (Y1998A) S phase cells determined by flow cytometry. Values represent means ± SD. e Representative live-cell imaging views of MEFs expressing mTurquoise-tagged PCNA to mark the boundaries of S phase by PCNA localization. One representative cell from each population of MEFs bearing either WT (top) or TTD-dead/Y1998A-mutated Bahcc1 (bottom) is shown. Bar, 10 μm. f, g Quantification of the total cell cycle length (f) and the length of the indicated cell cycle phases (g) based on live cell imaging. WT (blue), n = 40; TTD-dead/Y1998A-mutated (red), n = 35. Values represent means ± SD. Two-tailed unpaired t-test was conducted (P = 0.008). h, i Representative flow cytometry analysis (h) and quantification (i) of the chromatin-loaded PCNA levels in the S phase of HeLa cells with WT BAHCC1, BAHCC1 KO or the BAHCC1^TTD-defective mutation (Y1993A). The x-axis and y-axis in (h) measured the DNA content and chromatin-bound PCNA levels, respectively, with S phase cells showing in purple. S phase cells from three independent experiments were quantified in (i). One-tailed paired t-test was conducted using WT as controls (n = 3 biological replicates per group). *P ≤ 0.05; ***P ≤ 0.001. Values represent means ± SD. j A working model showing that H4K20me1:BAHCC1^TTD-mediated and H4K20me2:ORC1^BAH-mediated signaling axes promote the optimal MCM loading for efficient replication origin activation. Source data are provided as a Source Data file.