LRR1 knockout results in failure to disassemble replisomes and reduced rate of DNA replication. (A) Continuously cycling Cas9 cells were transfected with sgRNAs and fixed 1 d later, as in B–F and H. Sample immunofluorescence images are shown. Arrowheads indicate S-phase cells. Scale bar: 10 µm. Data are representative of four independent experiments. (B) Left: Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL). Paired Student’s t test; P = 5.5 × 10−4 (n = 4 independent experiments, n ≥ 4,272 cells per condition). Right: Abundance of chromatin-bound CDC45 in S-phase cells normalized to control cells. Paired Student’s t test; P = 1.1 × 10−3 (n = 4 independent experiments, n ≥ 4,272 cells per condition). (C) Rate of EdU incorporation in S-phase cells is plotted as a function of DNA content. Line plots are population medians in each bin; shaded error bars indicate 25th to 75th percentile (n ≥ 61 cells per bin, n = 9,393 and 6,182 cells total). Two-sample Student’s t test; ***, P < 1 × 10−3. Data are representative of four independent experiments. (D) Rate of EdU incorporation in early, mid, and late S-phase cells normalized to control cells. Early S: 2N–2.2N DNA, mid S: 2.9N–3.1N DNA, late S: 3.8N–4N DNA. Paired Student’s t test; P = 0.030, 6.2 × 10−4, 3.5 × 10−4 (n = 4 independent experiments, n ≥ 124 cells per condition). (E) Scatter plot of abundance of chromatin-bound CDC45 against DNA content in S-phase cells (R2 = 0.00 and 0.65; P = 0.98, P < 1 × 10−3; n = 3,000 random cells displayed). Data are representative of four independent experiments. (F) Top: Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + DMSO). Paired Student’s t test; P = 4.4 × 10−3, 0.015 (n = 3 independent experiments, n ≥ 4,993 cells per condition). Bottom: Abundance of chromatin-bound CDC45 in S-phase cells normalized to control cells. Paired Student’s t test; P = 0.031, 0.028 (n = 3 independent experiments, n ≥ 4,993 cells per condition). TRE, tetracycline-responsive element. (G) LRR1 was depleted in LRR1−/− cells expressing exogenous, doxycycline-inducible LRR1 by removing doxycycline and transfecting siRNA targeting exogenous LRR1. As a negative control, the same cell line was treated with doxycycline and nontargeting siRNA. Nuclear fluorescence signals were normalized to control cells (LRR1 positive). Paired Student’s t test; P = 5.9 × 10−4, 5.3 × 10−3, 3.5 × 10−3 (n = 3 independent experiments, n ≥ 1,041 cells per condition). (H) Abundance of chromatin-bound replisome components in S-phase cells is plotted as a function of DNA content. Line plots are population medians in each bin; shaded error bars indicate 25th to 75th percentile. Two-sample Student’s t test; ***, P < 1 × 10−3. CDC45: n ≥ 61 cells per bin, n ≥ 6,182 cells total. HA-GINS4: n ≥ 51 cells per bin, n ≥ 7,224 cells total. POLE2: n ≥ 71 cells per bin, n ≥ 4,647 cells total. Timeless: n ≥ 50 cells per bin, n ≥ 4,383 cells total. Data are representative of at least two independent experiments (HA-GINS4: two technical replicates). A.U., arbitrary unit; norm., normalized; R.F.U., relative fluorescence unit. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.