Fig 2: The MCM2–7 complex is required for cohesin loading during early S phase.(A) DAPI (blue), anti-Myc (red), and anti-MCM2 (green) staining of HeLa cells that stably expressed RAD21-Myc. Cells were transfected with the indicated siRNAs and arrested in early S phase with thymidine before fixation and staining. Scale bar, 5 μm. (B) Quantification of the chromatin intensities of RAD21-Myc of cells in (A). Each dot in the graph represents a single cell. Mean ± SD (siLuc, n = 184; siMCM2, n = 295; siNIPBL, n = 115). (C) Lysates of HeLa cells either mock transfected or transfected with siMCM2 were blotted with the indicated antibodies. (D) DAPI (blue), anti-Myc (red), and anti-TUBULIN (green) staining of telophase HeLa cells that stably expressed RAD21-Myc. Cells were transfected with the indicated siRNAs and released from nocodazole-mediated mitotic arrest for 4 hr before fixation. Scale bar, 5 μm. (E) Quantification of the RAD21-Myc chromatin intensities of cells in (D). Each dot in the graph represents a single cell. Mean ± SD (siLuc, n = 42; siMCM2, n = 42; siNIPBL, n = 38). (F) Log-phase HeLa cells and cells synchronized in early S phase by thymidine, G2 by the CDK1 inhibitor, or mitosis by nocodazole were collected and lysed in the presence of nuclease. The total cell lysates (input) and anti-MCM2 immunoprecipitate (IP) were blotted with the indicated antibodies. IgG IP from log-phase cells was used as a negative control. (G) HeLa cells were synchronized with thymidine for 16–18 hr, released from thymidine, and harvested at the indicated time points. Cells were lysed in the presence of nuclease. The total cell lysates (input) and anti-MCM2 IP were blotted with the indicated antibodies. (H) Flow cytometry analysis of log-phase HeLa cells and cells released from the thymidine arrest for the indicated times.

Fig 3: NIPBL and cohesin bind to MCM2–7 interdependently and co-localize in the genome.(A) HeLa cells were transfected with the indicated siRNAs, arrested in early S phase with thymidine, and lysed in the presence of nuclease. The total lysates (input) and anti-MCM2 IP were blotted with the indicated antibodies. (B) Venn diagrams showing the overlap of cohesin (RAD21)-occupied sites with those bound by NIPBL in two independent ChIP-seq experiments. (C) Region map showing that, at the majority of the NIPBL-occupied regions, the occupancy of RAD21 was detected reproducibly in the two ChIP-seq experiments.

Fig 4: DDK promotes the MCM–NIPBL–cohesin interaction.(A) DAPI (blue) and anti-Myc (red) staining of HeLa cells that stably expressed RAD21-Myc. Cells were transfected with the indicated siRNAs and arrested in early S phase with thymidine. Scale bar, 5 μm. (B) Lysates of HeLa cells transfected with the indicated siRNAs and synchronized in early S phase were treated with Turbo nuclease and immunoprecipitated with anti-MCM2. The total lysates (input) and anti-MCM2 immunoprecipitate (IP) were blotted with the indicated antibodies. (C) Lysates of HeLa cells were incubated with or without λPPase and blotted with the indicated antibodies. (D) Lysates of HeLa cells treated DMSO or the DDK kinase inhibitor XL413 (dissolved in DMSO) were blotted with the indicated antibodies. (E,F) DAPI (blue), anti-Myc (red), and anti-MCM2 (green) staining of RAD21-Myc-expressing HeLa cells that were treated with DMSO or XL413 and arrested in early S phase by thymidine. Scale bar, 5 μm. (G) HeLa cells were either transfected with the indicated siRNAs or treated with XL413, arrested in early S phase by thymidine, and lysed in the presence of Turbo nuclease. The total lysates (input) and anti-MCM2 IP were blotted with the indicated antibodies.

Fig 5: RPA promotes interphase sister-chromatid cohesion, cohesin loading in early S phase, and the MCM–NIPBL–cohesin interaction.(A) Representative images of G2-enriched HeLa cells transfected with the indicated siRNAs and stained with DAPI (blue in merge) and the early-replicating 466L19 FISH probe (red in merge). Selected paired FISH signals are magnified in inset. Scale bar, 5 μm. (B) Quantification of the distances between paired FISH signals of cells in (A). Mean ± SD (siLuc, n = 439; siSororin, n = 226; siRPA2, n = 59). (C) Quantification of the distances between paired FISH signals in G2-enriched HeLa cells transfected with the indicated plasmids and siRNAs. Mean ± SD (Vector + siLuc, n = 14; Vector + siRPA2, n = 78; Myc-RPA2 +siRPA2, n = 34). (D) DAPI (blue) and anti-Myc (red) staining of HeLa cells that stably expressed RAD21-Myc. Cells were transfected with the indicated siRNAs and arrested in early S phase with thymidine before fixation and staining. Scale bar, 5 μm. (E) Quantification of the chromatin intensities of RAD21-Myc of cells in (D). Mean ± SD (siLuc, n = 215; siMCM2, n = 298; siCDC7, n = 75; siNIPBL, n = 91; siSTAG2, n = 52; siRPA2, n = 131). (F,G) HeLa cells were transfected with the indicated siRNAs, synchronized in early S phase by thymidine, and lysed in the presence of Turbo nuclease. The total lysates (input) and anti-MCM2 immunoprecipitate (IP) were blotted with the indicated antibodies. (H) Lysates of HeLa cells transfected with the indicated siRNAs and arrested at early S phase with thymidine were blotted with the indicated antibodies.