Cohesin Can Remain Associated with Chromosomes during DNA Replication

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Cohesin Can Remain Associated with Chromosomes during DNA Replication James D.P. Rhodes, Judith H.I. Haarhuis, Jonathan B. Grimm, Benjamin D. Rowland, Luke D. Lavis, Kim A. Nasmyth Cell Reports Volume 20, Issue 12, Pages 2749-2755 (September 2017) DOI: 10.1016/j.celrep.2017.08.092 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 2749-2755DOI: (10.1016/j.celrep.2017.08.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 pcFRAP Permits Observation of Chromatin Binding over Long Time Periods (A) Immunoblot and in-gel fluorescence of Scc1-Halo and SNAP-H3.3 U2OS nuclear extract. (B) Live-cell microscopy images of Scc1-HaloJF549 and DY505SNAP-H3.3. Scale bar, 5 μm. (C) Schematic shows how residual fluorescent HaloTag ligand labels newly synthesized HaloTag fusion proteins. Incubation with an unlabeled ligand permanently blocks new proteins from becoming labeled. (D) Average intensity projections of z stacks from Scc1-Halo whole nuclear FRAP experiments. Scale bar, 5 μm. (E) Mean fluorescence intensity of Scc1-HaloJF549 nuclei 16 hr after bleaching of whole nucleus relative to prebleach intensity. Recovery was observed in the presence or absence of blocking HaloTag ligand. Data are represented as mean ± SEM. n = 11. (F) Graph depicting half nuclear FRAP for JF549SNAP-H3.3. n = 6. Data are represented as mean ± SEM. Cell Reports 2017 20, 2749-2755DOI: (10.1016/j.celrep.2017.08.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Characterization of Scc1-Halo WAPL1116-1119Δ Cell Line (A) Immunoblot of Scc1-Halo WAPL1116-1119Δ eGFP-PCNA U2OS nuclear extract. (B) Live-cell microscopy images of Scc1-HaloJF549 in wild-type or WAPL1116-1119Δ U2OS cells in interphase or mitosis. Scale bar, 5 μm. (C) Tracks of single Scc1-HaloJF549 molecules in wild-type or WAPL1116-1119Δ cells. (D) Histograms showing the number of molecules observed moving at different diffusion coefficients. Cell Reports 2017 20, 2749-2755DOI: (10.1016/j.celrep.2017.08.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Cohesin Remains Associated with the Same Area of Chromatin over Long Time Periods (A) Live-cell microscopy images of Scc1-HaloJF549 JF646SNAP-H3.3 WAPL1116-1119Δ U2OS cells before photobleaching with a 568-nm laser, immediately after photobleaching, and 23 hr later. Scale bar, 5 μm. Contrast enhanced in 23-hr time point. n = 10. (B) Fixed-cell microscopy images of Scc1-HaloJF549 WAPL1116-1119Δ U2OS cells in mitosis co-stained with an antibody against topoisomerase II. Scale bar, 2 μm. (C) Fluorescence line profiles of Scc1-HaloJF549 and topoisomerase II across metaphase 30 chromosomes from nine cells. Data are represented as mean ± SEM. Cell Reports 2017 20, 2749-2755DOI: (10.1016/j.celrep.2017.08.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Cohesin Can Remain Associated with DNA during S Phase Live-cell microscopy images of Scc1-HaloJF549 WAPL1116-1119Δ eGFP-PCNA cells in G1 before photobleaching, in G1 after photobleaching, in S phase, and in G2. It was not possible to image Scc1-HaloJF549 between G1 and G2 because of low starting signal in G1. This fluorescence was lost easily by photobleaching from eGFP and JF549 acquisition. Contrast has been enhanced in Scc1-HaloJF549 channel in post-bleach (after soluble fraction was bleached intentionally) and G2 (as there was a decrease in total nuclear signal). n = 12. Scale bar, 5 μm. See also Figures S3–S5. Cell Reports 2017 20, 2749-2755DOI: (10.1016/j.celrep.2017.08.092) Copyright © 2017 The Author(s) Terms and Conditions