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	Figure 1. Concentration- and time-of-addition-dependent effects of ICRF-193 on DNA replication in egg extracts. (A) Kinetoplast DNA decatenation activity of egg extract in the presence of 100 µM ICRF-193, or in the presence of the drug solvent (DMSO) alone, was assessed at the indicated times. Input (1) and decatenated (2) DNA molecules are indicated. (B) Same experiment as in (A), but in the presence of 10 µM ICRF-193. (C) Sperm nuclei were incubated in egg extract in the presence of DMSO or 100 µM ICRF-193, added at 0 min or at 20 min. Replication was monitored by [α-32P]-dATP incorporation. (D) Same experiment as in C but using 10 µM ICRF-193. (E) Percentage of replication in the presence of 100 µM ICRF-193 added at 0 min plotted against the control. Data are from 14 independent experiments. (F) Same as in (E), but ICRF-193 was added at 20 min. Data are from six independent experiments. (G) Sperm nuclei were incubated in egg extract in the presence or absence of 100 µM ICRF-193 added at the times indicated by the symbols in the insert. The percentage of replication in the presence of the drug is plotted against the control.
	Figure 2. Time-of-addition-dependent effects of ICRF-193 on nuclear structure and replication foci. (A) Sperm nuclei were incubated in egg extract in the presence of 20 µM rhodamin–dUTP and either drug solvent alone (DMSO) or 100 µM ICRF-193 added at 0 min. Reactions were stopped at the indicated times, fixed and stained with Hoechst. Replication foci were imaged as described in the ‘Materials and Methods’ section. Scale bar = 5 µm. (B) Mean and standard deviation (n = 30) of the number of replication foci per nucleus at 20 min and 30 min with or without ICRF-193. (C) Mean ± SD (n = 50) of nuclear area at 30, 45 and 60 min with DMSO or ICRF-193 added at 0 or 20 min. (D) Replication run-on assay. Sperm nuclei in egg extracts were pulsed for 5 min with rhodamin–dUTP at 40 or 90 min, fixed and stained with Hoechst. (E) Percentage of replicating nuclei at 40 or 90 min in the experiment shown in (D). In all, 150–200 nuclei were counted per sample.
	Figure 3. Alkaline gel electrophoretic analysis of ICRF-193-induced perturbation of DNA synthesis. (A) ICRF-193 added at 0 min, but not 20 min, reduces the abundance and perturbs the growth of nascent DNA strands. Sperm nuclei were incubated in egg extract containing [α-32P]-dATP in the presence of DMSO or 100 µM ICRF-193, added at 0 or at 20 min. Nascent strand abundance and growth was monitored by alkaline gel electrophoresis at the indicated time points. (B) Phosphorimager scan profiles of the 40 min lanes in (A). (C) Pulse-chase analysis of nascent strand growth. Sperm nuclei incubated in egg extract in the presence of DMSO or ICRF-193 added at 0 min or 20 min as indicated were labeled at 28 min with a 2 min pulse of [α-32P]dATP and chased for the indicated times with unlabeled dATP in the presence of roscovitin. Nascent strands were analyzed as in (A). (D) Nascent strand maturation analysis. Same experiment as in (A) except for the indicated times.
	Figure 4. DNA combing analysis of ICRF-193-induced perturbation of DNA synthesis. Sperm nuclei were incubated in egg extract plus biotin–dUTP for 35 or 45 min. DNA was extracted, combed on silanized coverslips and biotin-labeled replication eyes and total DNA were revealed using red and green fluorochrome-conjugated antibodies, respectively. (A) An exemplary DNA fiber visualized in the two channels and a merge picture. The midpoints of consecutive eyes are indicated by vertical white arrows. The distance between two consecutive arrows is referred to as eye-to-eye distance (ETED). (B) Representative single DNA molecules from the indicated samples and interpretative diagrams. Scale bar, 10 kb. Total replication extent (C), mean eye length (D), total fork density (E), intercluster distances (F) and distributions of ETED (size bins indicated in kb below horizontal axis) at 35 (G) and 45 min (H) were measured for each condition as described in the text.
	Figure 5. Effects of topo IIα depletion and addition on DNA replication kinetics and nuclear structure. (A and B) Topo IIα immunodepletion. Mock-depleted and topo IIα-depleted extracts were analyzed by western blotting with an anti-Xtopo IIα antibody (A; volume of extracts are indicated) and by kDNA decatenation (B). (C) Sperm nuclei were incubated in mock-depleted or topoIIα-depleted extracts containing [α-32P]-dATP plus or minus 100 µM ICRF-193 for 60 min, and the ratio of replicated DNA with and without ICRF-193 was calculated. (D) Sperm nuclei were incubated in mock-depleted (dotted lines) or topo IIα-depleted (solid lines) extracts containing [α-32P]-dATP supplemented with buffer (circles) or with recombinant human topo IIα (squares), and replication efficiency was measured at the indicated times. (E) Sperm nuclei were incubated in mock-depleted (upper panels) or topo IIα-depleted (lower panels) extracts in the presence of rhodamin–dUTP for 90 min, fixed and stained with Hoechst and observed under a fluorescence microscope. Scale bar, 2.5 µm. (F) Sperm nuclei were incubated in extracts containing [α-32P]-dATP supplemented with buffer (circles) or with recombinant human topo IIα (squares) added at 0 min, and replication was measured at the indicated times. (G) Same as in (F), except that buffer or topo IIα were added at 20 min.
	Figure 6. Gel electrophoretic and DNA combing analysis of replication intermediates synthesized in mock- and topoIIα-depleted extracts. (A) Pulse-chase analysis of nascent strand growth. Sperm nuclei incubated in mock- or topoIIα-depleted extracts were labeled at 50 min with a 10-min pulse of [α-32P]dATP and chased for the indicated times with unlabeled dATP in the presence of roscovitin. Nascent strands were electrophoresed in an alkaline agarose gel as in Figure 3A. (B–H) DNA combing analysis. Sperm nuclei were incubated in egg extract plus biotin–dUTP for 85, 105 or 115 min. DNA was extracted and combed as described in Figure 4. Total replication extent (B), total fork density (C), overall eye length (D) and mean intrafiber eye-to-eye distances (E) are shown for the three time points. To compare replication patterns within clusters, fibers subsets of comparable replication extents were selected and intrafiber eye lengths (F) and eye-to-eye distances (G) were measured. (H) Representative single DNA molecules from the indicated time samples and interpretative diagrams. Scale bar, 10 kb.
	Figure 7. Effects of topo IIα depletion (A and B) or addition (C and D) on chromatin binding of MCM3, MCM7, RecQ4, Cdc45 and topo IIα. Sperm nuclei were incubated in mock-depleted (A, dotted lines), topo IIα-depleted (A, solid lines) or undepleted (C) extracts added with recombinant htopo IIα (C, dark gray lines) or htopo IIα dilution buffer (C, light gray lines). (A and C) Purified chromatin at indicated times was analyzed by western blotting for the indicated proteins. Histone H3 was used as a loading control. Samples without egg extract (E-) or without sperm nuclei (S-) and 3 µl of unprocessed extract (E) were loaded as internal controls. The inset shown in (C) illustrates the electrophoretic resolution of human and Xenopus topo IIα. The signals quantified using Image J and normalized to histone H3 are shown as graphs. (B and D) Quantitation of MCM loading over multiple experiments and time points. Shown is the average ratio ± S.E.M of MCM signal (normalized to histone H3), in topo IIα-depleted (B) or htopo IIα-added (D) extracts, to MCM signal in control extracts. The observed ratios are significantly (****P < 0.10−4) different from the expected value of 1 in the null hypothesis, as indicated by asterisks and dotted lines.
	Figure 8. Effects of topo IIα inhibition by ICRF-193 on chromatin binding of MCM3, MCM7, RecQ4 and topo IIα. Sperm nuclei were incubated in undepleted extracts added with DMSO or 100 µM ICRF-193 at 0 or 20 min. (A) Purified chromatin at indicated times was analyzed by western blotting for the indicated proteins. Histone H3 was used as a loading control. Samples without egg extract (E-) or without sperm nuclei (S-) and 3 µl of unprocessed extract (E) were loaded as internal controls. (B) The signals quantified using Image J and normalized to histone H3 are shown as graphs. (C) Quantitation of MCM loading over multiple experiments and time points. Shown is the average ratio ± SEM of MCM signal (normalized to histone H3) in ICRF-193-treated extracts to MCM signal in control extracts. The observed ratios are not significantly different from the expected value of 1 in the null hypothesis.

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