Control experiments supporting the defective recycling model (related to Fig. 3). (A) Left: Sample immunofluorescence images of S-phase cells. In sgCNTL and sgLRR1 (CDC45^low) cells, EdU and CDC45 stains were largely colocalized, whereas the stains were less colocalized in sgLRR1 (CDC45^high) cells. Scale bar: 5 µm. Right: Colocalization of EdU and CDC45 stains was quantified in each nucleus using pixel-level Pearson’s correlation coefficient. Paired Student’s t test; sgCNTL vs. sgLRR1 (CDC45^low): P = 0.81; sgLRR1 (CDC45^low) vs. sgLRR1 (CDC45^high): P = 1.2 × 10⁻⁴; sgCNTL vs. sgLRR1 (CDC45^high): P = 3.5 × 10⁻³ (n = 3 independent experiments, n ≥ 1,230 cells per condition). (B) Schematic of experimental setup for subcellular fractionation (control experiment for Fig. 3, B–D). Serum-starved cells stably expressing Cas9 were transfected with sgRNAs and released from starvation in the presence of 10 µM aphidicolin to synchronize cells at the G1/S transition. (C) Control and LRR1 knockout cells were not released 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 C and replicates (relative to GAPDH or H4 loading controls) normalized to control cells. Paired Student’s t test; CDC45: P = 0.95, 0.70; GINS2: P = 0.87, 0.50; POLE1: P = 0.31, 0.60; Timeless: P = 0.25, 0.59; MCM2: P = 0.84, 0.38 (n = 4 independent experiments). (E) Subcellular localization of total CDC45, POLE2, Timeless, and MCM2 measured by immunofluorescence. Scale bar: 10 µm. Data are representative of three independent experiments. CDC45, POLE2, Timeless, and MCM2 were all exclusively nuclear localized, suggesting that the detergent-soluble fraction of these replisome components reflects the nucleoplasmic pool rather than the cytoplasmic pool. (F) Rate of EdU incorporation in S-phase cells was normalized to control cells (n ≥ 25,192 cells per condition). Data from n = 2 independent experiments. Expressing exogenous CDC45 or GINS4 alone did not rescue the DNA replication defect after LRR1 knockout. This result suggests that additional rate-limiting components or a combination of rate-limiting components is sequestered on chromatin, which is consistent with subcellular fractionation results where multiple replication factors were depleted from the soluble pool (Fig. 3 D). This led us to rescue DNA replication defects in LRR1 knockout cells by de-repressing E2F activity (Fig. 3 G). (G) Total nuclear levels of CDC45, Timeless, and POLE2 were measured in S-phase cells and normalized to control cells. Paired Student’s t test; CDC45: P = 0.057, 1.4 × 10⁻³, 1.2 × 10⁻³ (n = 4 independent experiments, n ≥ 9,916 cells per condition); Timeless: P = 0.029, 1.3 × 10⁻³, 2.4 × 10⁻³ (n = 3 independent experiments, n ≥ 10,680 cells per condition); POLE2: P = 0.356, 0.032, 0.060 (n = 3 independent experiments, n ≥ 2,532 cells per condition). (H) Rate of EdU incorporation in S-phase cells normalized to control cells (sgCNTL + siCNTL). Cells were categorized as CDC45^low (unsuccessful knockout) or CDC45^high (successful knockout) based on the abundance of chromatin-bound CDC45. Paired Student’s t test; P = 0.069, 0.31, 0.015 (n = 4 independent experiments, n ≥ 164 cells per condition). KO, knockout; norm., normalized; TRE, tetracycline-responsive element. In all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001.