Figure 3.
Failure to disassemble replisomes after LRR1 loss suppresses DNA replication by blocking the recycling of replication factors. (A) Continuously cycling Cas9 cells were transfected with sgRNAs and fixed 1 d later. Scatter plot of rate of EdU incorporation against abundance of chromatin-bound CDC45 in S-phase cells (n = 3,000 random cells displayed). Left: R2 = 0.38, P < 1 × 10−3. Right: Shaded area represents cells with high chromatin-bound CDC45, where R2 = −0.47, P < 1 × 10−3. Data are representative of four independent experiments. (B) Left: Experimental setup. Right: Distribution of DNA content (n ≥ 17,152 cells per condition). (C) Cells were released for 5 h from an aphidicolin block and harvested. Detergent-soluble (S) and insoluble (I) proteins were fractionated and immunoblotted. Soluble and insoluble fractions were loaded at a ratio of 1:2. Data are representative of four independent experiments. (D) Quantification of Western blots (relative to GAPDH or H4 loading controls) normalized to control cells. Paired Student’s t test; CDC45: P = 0.045, 7.1 × 10−4; GINS2: P = 0.15, 0.014; POLE1: P = 0.011, 0.040; Timeless: P = 0.014, 7.0 × 10−3; and MCM2: P = 0.53, 0.78 (n = 4 independent experiments). (E) Left: Schematic of DNA fiber assay. LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1. Right: Sample images from DNA fiber assay. (F) Quantification of DNA fiber assay. Left: Percentage of total labeled structures that are replication origins. Two-sample Student’s t test; P = 1.9 × 10−3 (n = 3 independent experiments, n ≥ 28 labeled structures per condition). Right: Ratio of IdU/CldU track lengths. Box plots represent the interquartile range (IQR), while whiskers represent the nonoutlier minimum and maximum; outliers are values that are more than 1.5 × IQR away from box edges. Two-sample Student’s t test, P = 0.80; two-sample Kolmogorov-Smirnov test, P = 0.51 (data pooled from n = 3 independent experiments, n = 85 and 70 labeled tracks per condition). (G) Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + siCNTL). Cells were categorized as CDC45low (unsuccessful knockout) or CDC45high (successful knockout) based on the abundance of chromatin-bound CDC45. Paired Student’s t test; P = 0.10, 0.77, 0.011 (n = 4 independent experiments, n ≥ 198 cells per condition). (H) Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + siCNTL). Paired Student’s t test; P = 0.10, 0.27, 0.025 (n = 2 independent experiments, n ≥ 25 cells per condition). A.U., arbitrary unit; R.F.U., relative fluorescence unit. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Failure to disassemble replisomes after LRR1 loss suppresses DNA replication by blocking the recycling of replication factors. (A) Continuously cycling Cas9 cells were transfected with sgRNAs and fixed 1 d later. Scatter plot of rate of EdU incorporation against abundance of chromatin-bound CDC45 in S-phase cells (n = 3,000 random cells displayed). Left: R2 = 0.38, P < 1 × 10−3. Right: Shaded area represents cells with high chromatin-bound CDC45, where R2 = −0.47, P < 1 × 10−3. Data are representative of four independent experiments. (B) Left: Experimental setup. Right: Distribution of DNA content (n ≥ 17,152 cells per condition). (C) Cells were released for 5 h from an aphidicolin block and harvested. Detergent-soluble (S) and insoluble (I) proteins were fractionated and immunoblotted. Soluble and insoluble fractions were loaded at a ratio of 1:2. Data are representative of four independent experiments. (D) Quantification of Western blots (relative to GAPDH or H4 loading controls) normalized to control cells. Paired Student’s t test; CDC45: P = 0.045, 7.1 × 10−4; GINS2: P = 0.15, 0.014; POLE1: P = 0.011, 0.040; Timeless: P = 0.014, 7.0 × 10−3; and MCM2: P = 0.53, 0.78 (n = 4 independent experiments). (E) Left: Schematic of DNA fiber assay. LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1. Right: Sample images from DNA fiber assay. (F) Quantification of DNA fiber assay. Left: Percentage of total labeled structures that are replication origins. Two-sample Student’s t test; P = 1.9 × 10−3 (n = 3 independent experiments, n ≥ 28 labeled structures per condition). Right: Ratio of IdU/CldU track lengths. Box plots represent the interquartile range (IQR), while whiskers represent the nonoutlier minimum and maximum; outliers are values that are more than 1.5 × IQR away from box edges. Two-sample Student’s t test, P = 0.80; two-sample Kolmogorov-Smirnov test, P = 0.51 (data pooled from n = 3 independent experiments, n = 85 and 70 labeled tracks per condition). (G) Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + siCNTL). Cells were categorized as CDC45low (unsuccessful knockout) or CDC45high (successful knockout) based on the abundance of chromatin-bound CDC45. Paired Student’s t test; P = 0.10, 0.77, 0.011 (n = 4 independent experiments, n ≥ 198 cells per condition). (H) Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + siCNTL). Paired Student’s t test; P = 0.10, 0.27, 0.025 (n = 2 independent experiments, n ≥ 25 cells per condition). A.U., arbitrary unit; R.F.U., relative fluorescence unit. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal