Analysis of replisome disassembly and DNA replication after LRR1 loss (related to Fig. 2). (A) Nuclear abundance of chromatin-bound (histone H2B-mTurquoise) and nonchromatin-bound (hDHB-mVenus; see Fig. S5 C) fluorescent proteins in cells that were directly fixed and in cells that were preextracted before fixation. Two-sample Student’s t test; P = 8.8 × 10−3, 2.2 × 10−4 (n = 2 technical replicates, n ≥ 14,386 cells per replicate). (B) Cell-cycle progression in asynchronously cycling cells that were transfected with sgRNAs and fixed 1 d later. Left: G1/S/G2 gating using EdU-DNA content scatter plot (n = 2,000 random cells displayed) and distribution of DNA content (n ≥ 20,625 cells per condition). Top right: quantification of G1/S/G2 gating from scatter plot. Bottom right: quantification of G1/S/G2 gating in n = 4 independent experiments, shown as mean ± SD. P values are from paired Student’s t tests (n ≥ 19,134 cells per condition). To reconcile the fact that (i) percentages of G1/S/G2 cells are not significantly different between control and LRR1 knockout cells and (ii) there is indeed G2 arrest in these cells (lower percentage of cells that are going into mitosis, as shown in Fig. 4), note that these scatter plots are from cells fixed 1 d after LRR1 knockout, and this is likely the first cell cycle that the cells went through in the absence of LRR1 protein. As a result, the G2 arrest is only beginning to manifest and is not captured by a higher percentage of G2 cells. (C) RPE-1 hTERT cells stably expressing Cas9 were transfected with sgRNAs and fixed 1 d later. Left: Rate of EdU incorporation in S-phase cells as a function of DNA content. Right: Abundance of chromatin-bound CDC45 in S-phase cells as a function of DNA content. Line plots are population medians in each bin; shaded error bars indicate 25th to 75th percentile (n ≥ 68 cells per bin, n = 36,413 and 19,133 cells total). Two-sample Student’s t test; ***, P < 1 × 10−3. Data are representative of two technical replicates. (D) Cells were treated with a p97 inhibitor (CB-5083, 4 µM) for 2 h and fixed. Left: Rate of EdU incorporation in S-phase cells as a function of DNA content. Right: Abundance of chromatin-bound CDC45 in S-phase cells as a function of DNA content. Line plots are population medians in each bin; shaded error bars indicate 25th to 75th percentile (n ≥ 78 cells per bin, n = 3,985 and 4,507 cells total). Two-sample Student’s t test; ***, P < 1 × 10−3. Data are representative of two independent experiments. (E) Cells expressing CRISPR-resistant LRR1 from a doxycycline-inducible promoter were transfected with sgCNTL or sgLRR1 and selected for stable single-cell clones. Cells were cultured in doxycycline to maintain expression of exogenous LRR1. Rate of EdU incorporation was measured in S-phase cells after replacing doxycycline with DMSO or doxycycline for 1 d. Two-sample Student’s t test; P = 0.29, 3.0 × 10−3, 0.030 (n = 2 technical replicates, n ≥ 1,436 cells per replicate). The reduction in DNA replication rate was mild after the removal of doxycycline, possibly due to incomplete transcriptional shutdown from the doxycycline-inducible promoter. To further deplete LRR1, LRR1−/− cells expressing exogenous LRR1 were transfected with LRR1-targeting siRNA along with doxycycline removal, which achieved near complete (∼97%) depletion of LRR1 protein (Fig. S2 F) and a large reduction in DNA replication rate and failure to unload CMG helicases from chromatin in S-phase cells (Fig. 2 G, middle and bottom). (F) LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1 by removing doxycycline and transfecting siRNA targeting exogenous LRR1 1 d before fixation. As a negative control, the same cell line was treated with doxycycline and nontargeting siRNA. Cells were harvested and blotted for LRR1. Arrowhead indicates LRR1-specific band; the other band is a nonspecific band. Right: Quantification of Western blots (relative to actin loading control) normalized to control. Paired Student’s t test; P = 8.3 × 10−6 (n = 3 independent experiments). (G) Abundance of chromatin-bound replisome components in S-phase cells normalized to control cells. Higher levels of CDC45, POLE2, Timeless, and HA-GINS4 were detected in LRR1 knockout cells. Paired Student’s t test; P = 0.032, 0.024, 0.21, 0.27, 0.31, 0.028, 0.040, 0.020 (n = 2 independent experiments, n ≥ 3,931 cells per condition; HA-GINS4: n = 2 technical replicates, n ≥ 3,552 cells per replicate). A.U., arbitrary unit; norm., normalized; R.F.U., relative fluorescence unit; TRE, tetracycline-responsive element. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure S2.

Analysis of replisome disassembly and DNA replication after LRR1 loss (related to Fig. 2). (A) Nuclear abundance of chromatin-bound (histone H2B-mTurquoise) and nonchromatin-bound (hDHB-mVenus; see Fig. S5 C) fluorescent proteins in cells that were directly fixed and in cells that were preextracted before fixation. Two-sample Student’s t test; P = 8.8 × 10−3, 2.2 × 10−4 (n = 2 technical replicates, n ≥ 14,386 cells per replicate). (B) Cell-cycle progression in asynchronously cycling cells that were transfected with sgRNAs and fixed 1 d later. Left: G1/S/G2 gating using EdU-DNA content scatter plot (n = 2,000 random cells displayed) and distribution of DNA content (n ≥ 20,625 cells per condition). Top right: quantification of G1/S/G2 gating from scatter plot. Bottom right: quantification of G1/S/G2 gating in n = 4 independent experiments, shown as mean ± SD. P values are from paired Student’s t tests (n ≥ 19,134 cells per condition). To reconcile the fact that (i) percentages of G1/S/G2 cells are not significantly different between control and LRR1 knockout cells and (ii) there is indeed G2 arrest in these cells (lower percentage of cells that are going into mitosis, as shown in Fig. 4), note that these scatter plots are from cells fixed 1 d after LRR1 knockout, and this is likely the first cell cycle that the cells went through in the absence of LRR1 protein. As a result, the G2 arrest is only beginning to manifest and is not captured by a higher percentage of G2 cells. (C) RPE-1 hTERT cells stably expressing Cas9 were transfected with sgRNAs and fixed 1 d later. Left: Rate of EdU incorporation in S-phase cells as a function of DNA content. Right: Abundance of chromatin-bound CDC45 in S-phase cells as a function of DNA content. Line plots are population medians in each bin; shaded error bars indicate 25th to 75th percentile (n ≥ 68 cells per bin, n = 36,413 and 19,133 cells total). Two-sample Student’s t test; ***, P < 1 × 10−3. Data are representative of two technical replicates. (D) Cells were treated with a p97 inhibitor (CB-5083, 4 µM) for 2 h and fixed. Left: Rate of EdU incorporation in S-phase cells as a function of DNA content. Right: Abundance of chromatin-bound CDC45 in S-phase cells as a function of DNA content. Line plots are population medians in each bin; shaded error bars indicate 25th to 75th percentile (n ≥ 78 cells per bin, n = 3,985 and 4,507 cells total). Two-sample Student’s t test; ***, P < 1 × 10−3. Data are representative of two independent experiments. (E) Cells expressing CRISPR-resistant LRR1 from a doxycycline-inducible promoter were transfected with sgCNTL or sgLRR1 and selected for stable single-cell clones. Cells were cultured in doxycycline to maintain expression of exogenous LRR1. Rate of EdU incorporation was measured in S-phase cells after replacing doxycycline with DMSO or doxycycline for 1 d. Two-sample Student’s t test; P = 0.29, 3.0 × 10−3, 0.030 (n = 2 technical replicates, n ≥ 1,436 cells per replicate). The reduction in DNA replication rate was mild after the removal of doxycycline, possibly due to incomplete transcriptional shutdown from the doxycycline-inducible promoter. To further deplete LRR1, LRR1−/− cells expressing exogenous LRR1 were transfected with LRR1-targeting siRNA along with doxycycline removal, which achieved near complete (∼97%) depletion of LRR1 protein (Fig. S2 F) and a large reduction in DNA replication rate and failure to unload CMG helicases from chromatin in S-phase cells (Fig. 2 G, middle and bottom). (F) LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1 by removing doxycycline and transfecting siRNA targeting exogenous LRR1 1 d before fixation. As a negative control, the same cell line was treated with doxycycline and nontargeting siRNA. Cells were harvested and blotted for LRR1. Arrowhead indicates LRR1-specific band; the other band is a nonspecific band. Right: Quantification of Western blots (relative to actin loading control) normalized to control. Paired Student’s t test; P = 8.3 × 10−6 (n = 3 independent experiments). (G) Abundance of chromatin-bound replisome components in S-phase cells normalized to control cells. Higher levels of CDC45, POLE2, Timeless, and HA-GINS4 were detected in LRR1 knockout cells. Paired Student’s t test; P = 0.032, 0.024, 0.21, 0.27, 0.31, 0.028, 0.040, 0.020 (n = 2 independent experiments, n ≥ 3,931 cells per condition; HA-GINS4: n = 2 technical replicates, n ≥ 3,552 cells per replicate). A.U., arbitrary unit; norm., normalized; R.F.U., relative fluorescence unit; TRE, tetracycline-responsive element. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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