Fig 2: Pola1 facilitates parental histone H3.3 recycling.a Representative live-cell images of parental histones H3.3-SNAP signals at the indicated time points in WT (n = 61), Pole4 KO (n = 66), Mcm2-2A (n = 66), Pola1-2A (n = 74) mES single and Mcm2-2A Pole4 KO double mutant (n = 72) cell lines. Purple channels are 647-SiR labeled-parental H3.3-SNAP, green channels are for mAG tagged-geminin expression. Scale bar, 10 µm. Tracked cells were circled. b The average time from G1/S to mitosis for WT, Pole4 KO, Mcm2-2A, Pola1-2A, and Mcm2-2A Pole4 KO double mutant mES cell lines based on the appearance of mAG tagged-geminin (G1/S) and the reduction of mAG tagged-geminin signals (mitosis). c–g Quantification of H3.3-SNAP signals at G1/S and G2 of each mother cell and G1 of its two daughter cells in WT, Pole4 KO, Mcm2-2A, Pola1-2A, and Mcm2-2A + Pole4 KO mutant mES cell lines. The cell cycle stage of each cell was based on mAG tagged-geminin signals. h Comparison of the relative amount of parental H3.3-SNAP at individual cells at late G2 in WT, Pole4 KO, Mcm2-2A, Pola1-2A and Mcm2-2A + Pole4 KO mES cell lines. The center line, the box limits and the whiskers were defined as described in Fig. 2c. b–g Data are presented as means ± SD. a–h n number of cells from two independent experiments. c–h Statistical analysis was performed by two-tailed unpaired Student t test with the P values marked on the graphs, ns no significant difference.

Fig 3: Histone chaperones Mcm2 and Pole4 are involved in the recycling of parental H3.1.a Upper: an experimental scheme for the analysis of H3.1-SNAP in mES cells using live-cell imaging. Lower: representative live-cell images of TMR of parental histone H3.1-SNAP at the indicated time points in WT (n = 63), Pole4 KO (n = 72), Mcm2-2A (n = 66) and Pole4 KO + Mcm2-2A double mutant (n = 67) mES cells. Scale bar, 10 µm. Two daughter cells arising from the mother cell were circled. b Quantification of parental H3.1-SNAP signals WT, Pole4 KO, Mcm2-2A and Mcm2-2A + Pole4 KO mES cells at G1/S and G2 of each mother cell and G1 of the two individual daughter cells. Data are presented as means ± SD. c Boxplot of relative parental H3.1-SNAP signals at G2 calculated in (b) among WT, Pole4 KO, Mcm2-2A and Mcm2-2A + Pole4 KO mES cell lines. The center line is the medians of all data points, with the limits corresponding to the upper and the lower quartiles, respectively, and the whiskers representing the largest and smallest values. a–c n number of cells from two independent experiments. b, c Statistical analysis was performed by two-tailed unpaired Student t test with P values shown on the graphs. ns no significant difference.

Fig 4: Mcm2-2A, Pole3 KO, and Pole4 KO mutants defective in parental histone transfer show abnormal chromosome segregation.a Deletion of either H3f3a or H3f3b results in defects in mitosis. Representative live-cell images of H3.1/H3.3-SNAP at the indicated time points (20 min before (-) and after (+) anaphase) in WT, H3f3a KO and H3f3b KO mES cells (n > 160). Nuclear abnormalities marked by arrows including chromosome bridges, misaligned chromosomes and lagging chromosomes observed in anaphase was counted in H3f3a KO/H3f3b KO mES cells. Scale bar, 10 µm. b Percentages of abnormal mitotic cells in WT, H3f3a KO and H3f3b KO mES cells. Images at anaphase were used for quantification. c Representative live-cell images of H3.3-SNAP signals collected at indicated time (20 min before (-) and after (+) anaphase) in WT, Pole3 KO, Pole4 KO, Mcm2-2A, Mcm2-2A + Pole3 KO, Mcm2-2A + Pole4 KO, Hira KO, and Daxx KO mES cells (n > 170). The nuclear abnormalities mentioned in (a) were observed in Pole3 KO, Pole4 KO, Mcm2-2A, Mcm2-2A + Pole3 KO, Mcm2-2A + Pole4 KO, and Daxx KO mES cells. Defects are indicated by arrows. Scale bar, 10 µm. d Percentages of abnormal mitotic cells in WT, Pole3 KO, Pole4 KO, Mcm2-2A, Mcm2-2A + Pole3 KO, Mcm2-2A + Pole4 KO, Hira KO, and Daxx KO cell lines. Images at anaphase were used for quantification. b, d Data are presented as means ± SD. Statistical analysis was performed by two-tailed unpaired Student t test with the P values shown on the graphs (ns, no significant difference). a–d n > 160 cells from three independent experiments were analyzed. e The percentage of G2/M phase cells increases in WT, Pole3 KO, Pole4 KO, Mcm2-2A, single, Mcm2-2A + Pole3 KO, and Mcm2-2A Pole4 KO double mutant cell lines. Cells were collected for flow cytometry analysis of DNA content, and the percentage of cells at G2/M was calculated. Data are presented as means ± SD. Statistical analysis was performed by two-tailed unpaired Student t test, and the P values were marked on the graphs (ns no significant difference). Four independent replicates were conducted for each cell line.

Fig 5: Mcm2, Pole3, and Pole4 are required for faithfully parental histone H3.3 recycling during S phase.a Representative live-cell images of parental histones H3.3-SNAP signals at the indicated time points in WT (n = 74), Pole3 KO (n = 80), Pole4 KO (n = 66), and Mcm2-2A (n = 69) mES single mutant cell lines. Scale bar, 10 µm. b Quantification of H3.3-SNAP signals at each individual cell of WT, Pole3 KO, Pole4 KO, and Mcm2-2A single mutant mES cells. Data are presented as means ± SD. c Comparison of the relative amount of parental H3.3-SNAP at individual cells at late G2 in WT, Pole3 KO, Pole4 KO, Mcm2-2A, Mcm2-2A + Pole3 KO, and Mcm2-2A + Pole4 KO mES cell lines. The center line, the box limits and the whiskers are defined as described in Fig. 2c. a–c n number of cells from two independent experiments. b, c Two-tailed unpaired Student t tests were performed with the P values marked on the graphs, and ns no significant difference.