Shintomi K , Hirano T .

18H02381 MEXT | Japan Society for the Promotion of Science (JSPS), 19H05755 MEXT | Japan Society for the Promotion of Science (JSPS), 20H05938 MEXT | Japan Society for the Promotion of Science (JSPS), 15H05971 MEXT | Japan Society for the Promotion of Science (JSPS), 18H05276 MEXT | Japan Society for the Promotion of Science (JSPS)

	Fig. 1: Manipulation of buffer conditions in the mitotic chromatid reconstitution assay. a, b Xenopus sperm nuclei were mixed with a cocktail of six purified proteins (topo IIα, condensin I, a truncated version of histone H2A.XF-H2B dimer, Npm2, Nap1, and FACT) in a buffer containing no MgCl2 or increasing concentrations of MgCl2. The concentrations of other chemical ingredients in the buffer are listed in Table 1. After a 150-min incubation at 22 °C, the resultant structures were fixed and labeled with antibodies against topo IIα and CAP-G (a subunit of condensin I). DNA was counterstained with DAPI (a). Integral signal intensities of topo IIα or CAP-G were divided by those of DAPI in the same segmented region, and the values normalized to the average value in the reaction containing 5.0 mM MgCl2 are plotted. The mean ± s.d. is shown (n = 9 clusters of chromatin). P values were assessed by two-tailed Welch’s t-test (ns, not significant) (b). c–e KCl concentrations were titrated in the reconstitution assay. Immunolabeling and signal quantification were carried out as above (c, e). The perimeter of a DAPI-positive segmented area was divided by the integral intensity of DAPI in the same area, and the resultant values are plotted as individualization indices (d). The mean ± s.d. is shown. The sample sizes (n, the number of clusters of chromatin) are 10 (50, 80, and 120 mM KCl) and 8 (200 mM KCl). P values were assessed by two-tailed Welch’s t-test (ns, not significant) (d, e). f–h ATP concentrations were titrated in the reconstitution assay (f). Individualization indices and signal intensities were analyzed as above. The mean ± s.d. is shown. The sample sizes (n, the number of clusters of chromatin) are 12 (0 and 0.10 mM ATP), 17 (0.30 mM ATP) and 20 (1.0 mM KCl). P values were assessed by a two-tailed Welch’s t-test. ns not significant (g, h). Bars, 5 µm.
	Fig. 2: Topo IIα-ΔCTD is proficient in chromatid individualization but is deficient in chromatid thickening. a Schematic presentation of structures of full-length (FL) and CTD-deleted (ΔCTD) versions of recombinant Xenopus laevis topo IIα. b Purified topo IIα-FL and topo IIα-ΔCTD were analyzed by SDS-PAGE and stained with Coomassie Blue. The same set of samples was also subjected to immunoblotting using anti-FLAG antibodies. This experiment was repeated three times with similar results. c–e Xenopus sperm nuclei were incubated in the reconstitution reaction mixture containing either topo IIα-FL or topo IIα-ΔCTD. At the indicated time points, the resultant chromatin was fixed and stained with DAPI. Blow-up images of cropped parts (indicated by the dashed rectangles in the original 120-min images) are shown on the right (c). Individualization indices at 120 min are plotted. The mean ± s.d. is shown (n = 12 clusters of chromatids). P values were assessed by two-tailed Welch’s t-test (ns not significant) (d). Profiles of normalized signal intensities of DAPI along lines drawn perpendicular to chromatid axes were analyzed. The mean ± s.d. is shown (n = 15 lines from 5 chromatids) (e). f, g Chromatid reconstitution assays were performed with topo IIα-FL or topo IIα-ΔCTD in a buffer containing 80 mM or 150 mM KCl. After a 150-min incubation, the resultant structures were fixed and processed for immunolabeling (f). Signal intensities of topo IIα and CAP-G on chromatids were analyzed. The mean ± s.d. is shown (n = 9 clusters of chromatin). P values were assessed by two-tailed Welch’s t-test (ns not significant) (g). Bars, 5 µm.
	Fig. 3: Topo IIα-ΔCTD is proficient in DNA decatenation but is deficient in DNA catenation a, b Catenated DNA (100 ng) was mixed with a low amount (40 ng) of topo IIα-FL or topo IIα-ΔCTD in a buffer containing 2 mM ATP, 5 mM MgCl2, and either 80 mM or 150 mM KCl. After incubation at 22 °C, the resultant DNAs were recovered at the indicated time points, purified, and analyzed by agarose gel electrophoresis. The gel was stained with ethidium bromide (a). Intensities of decatenated DNA were quantified. The mean ± s.d. from three independent experiments is shown (b). c, d Nicked circular DNA (100 ng) was mixed with an excess amount (400 ng) of topo IIα-FL or topo IIα-ΔCTD in a buffer containing 5 mM MgCl2, and either 80 mM or 150 mM KCl. After a 10-min incubation at 22 °C, the reactions were supplemented with 2 mM AMP-PNP and incubated for another 20 min. The DNAs were then purified and analyzed by agarose gel electrophoresis (c). Intensities of catenated and knotted DNAs were quantified. In each experiment, values of the topo IIα-ΔCTD reaction were normalized to those of the topo IIα-FL reaction. The mean ± s.d. from three independent experiments is shown (d). e Chromatid reconstitution assays were performed with topo IIα-FL or topo IIα-ΔCTD in a buffer containing 80 mM KCl. After a 150-min incubation at 22 °C, the mixtures were diluted by adding the same volume of reaction mixtures containing 80 mM KCl or 220 mM KCl so that the final concentrations of KCl became 80 mM or 150 mM. After another 20-min incubation at 22 °C, the resultant chromosomes were fixed and processed for immunolabeling. Blow-up images of cropped parts (indicated by the dashed rectangles in the merged images) are shown in grayscale. This experiment was repeated three times with similar results. Bar, 5 µm.
	Fig. 4: An unusual chromatin structure is produced in the cell-free extract depleted of both topo IIα and Asf1. a, b Mouse sperm nuclei were incubated in a control extract (Δmock) or an extract depleted of both topo IIα and Asf1 (Δtopo IIα ΔAsf1) for 150 min and labeled with antibodies against topo IIα and histone H3. DNA was counterstained with DAPI (a). The compaction indices (the average DAPI intensities per unit area) are plotted. The mean ± s.d. is shown (n = 10 clusters of chromatin). P values were assessed by two-tailed Welch’s t-test (b). c, d Mouse sperm nuclei were incubated in the Δtopo IIα ΔAsf1 extract at 22 °C. At the indicated time points, the reaction mixtures were fixed and labeled with antibodies against CAP-G (a condensin I subunit) and CAP-H2 (a condensin II subunit). DNA was counterstained with DAPI. This experiment was repeated three times with similar results (c). Blow-up images of the cropped part (indicated by the dashed rectangle in the original DAPI image at 150 min) are shown in grayscale (DAPI) and pseudo-colors (merged images for the indicated combinations) (d). Bars, 5 µm.
	Fig. 5: Topo IIα-FL, but not topo IIα-ΔCTD, can efficiently rescue the defects observed in the extracts depleted of both topo IIα and Asf1. a, b Mouse sperm nuclei were incubated in a control extract (Δmock) or an extract depleted of either topo IIα (Δtopo IIα), Asf1 (ΔAsf1), or both (Δtopo IIα ΔAsf1). After a 150-min incubation at 22 °C, the resultant structures were labeled with antibodies against the linker histone H1.8 and CAP-G. DNA was counterstained with DAPI. This experiment was repeated three times with similar results (a). Blow-up images of the cropped part (indicated by the dashed rectangle in the original DAPI image in a Δtopo IIα ΔAsf1 extract) are shown in grayscale (DAPI) and pseudo-colors (merged images for the indicated combinations) (b) c, d An extract depleted of topo IIα and Asf1 (Δtopo IIα ΔAsf1) was supplemented with either buffer, topo IIα-FL, or topo IIα-ΔCTD. After a 30-min incubation at 22 °C, mouse sperm nuclei were added to these extracts and incubated for another 150 min. The resultant chromatin structures were fixed and processed for immunolabeling with the antibodies indicated (c). The compaction indices were analyzed and are shown in Fig. 4b. The mean ± s.d. is shown (n = 12 clusters of chromatin). P values were assessed by two-tailed Welch’s t-test (d). e, f Mouse sperm nuclei were first incubated in a Δtopo IIα ΔAsf1 extract to allow sparkler formation. At 150 min, the reaction mixtures were supplemented with either buffer, topo IIα-FL, or topo IIα-ΔCTD. After another 150-min incubation at 22 °C, the samples were fixed and processed for immunolabeling (e). The compaction indices were analyzed and are shown as above. The mean ± s.d. is shown (n = 12 clusters of chromatin). P values were assessed by two-tailed Welch’s t-test (f). Bars, 5 µm.
	Fig. 6: Models for the actions of topo IIα in crowded environments created during mitotic chromatid assembly. a A model for chromatid thickening by collaborative actions of topo IIα and condensins. At the initial step of chromatid assembly, intra-chromatid entanglements are hardly generated because loops are distantly located from one another (step 1). As condensins extrude the loops, the distance between neighboring loops gets shortened (step 2), allowing chromatin-associated topo IIα to generate entanglements between loops (step 3: intra-chromatid entanglement). The resultant intra-chromatid entanglements in turn stabilize chromatid axes and may promote further extrusion of the loops and thickening of the chromatids (step 4). For simplicity, structural changes of an individualized, single chromatid are shown here. b A model for resolution of a pre-assembled sparkler. A sparkler is composed of multiple non-nucleosomal DNAs that are heavily entangled with each other and bound by the linker histone H1.8 (step 1). When topo IIα is added into the reaction, its CTD competes with the linker histone for the non-nucleosomal DNAs and delivers the enzymatic core into the inside of the sparkler (step 2). Topo IIα catalyzes disentanglement reactions there (step 3: inter-chromatid disentanglement), which in turn promotes further dissociation of the linker histone and resolves the sparkler (step 4). For simplicity, condensins’ actions are not depicted here.

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