Gardner NJ , Gillespie PJ , Carrington JT , Shanks EJ , McElroy SP , Haagensen EJ , Frearson JA , Woodland A , Blow JJ .

Wellcome Trust , 14301 Cancer Research UK

             DNA replication
             cell cycle

	Graphical Abstract
	Figure 1. A Cell-Based Screen for Licensing Inhibitors (A) Asynchronous U2OS cells were pulsed with EdU for 30 min, then labeled for EdU incorporation, chromatin-bound MCM2, and total DNA, and analyzed by flow cytometry. For the plot, G1 cells (G1 DNA content, EdU negative) were colored red, S-phase cells (EdU positive) were colored blue, and G2 cells (G2 DNA content, EdU negative) were colored orange, and then plotted for total DNA content and chromatin-bound MCM2. (B) Outline of the protocol for assaying potential licensing inhibitors. (C and D) Immunofluorescence images of cells after the RO3306 block (C) or 8 hr later at the end of the assay (D) immunostained for MCM4 (green) and with DAPI for DNA (blue). Scale bar, 10 μm. (E) Cartoon of possible outcomes of the screen derived from total cellular DNA content and amount of chromatin-bound MCM2–7. (F) Examples of output from the cell-based screen: (i) late S/G2 enriched starting cells; (ii) non-specific inhibition, showing G2 accumulation; (iii) no inhibition, showing licensed G1 cells; (iv) hit compound showing unlicensed G1 cells. See also Figures S1 and S6.
	Figure 2. Licensing Assays in Human and Xenopus Systems (A) Percentage inhibition of MCM4 loading observed with all 24,000 compounds in the initial screen. (B) Validated hits from the primary and secondary screen in U2OS cells were assayed for their ability to inhibit an in vitro licensing assay in Xenopus egg extracts. The degree of licensing is expressed as a percentage of that observed in control “Licensing Factor” extract. (C) Immunoblot of total and chromatin-bound MCM2, MCM3, and MCM5 in U2OS cells treated with either RL5a or DMSO. (D) Immunoblot of chromatin-bound MCM7 from Xenopus “Licensing Factor” extract treated with DMSO, geminin, RL5a, RL5b, or RL5c. (E) Structures of RL5a–e. See also Figures S2 and S6.
	Figure 3. Activity of RL5a in Whole Xenopus Egg Extract (A) Sperm nuclei were incubated in whole Xenopus egg extract supplemented with 25, 50, 100, 150 or 200 μM RL5a, DMSO, or geminin. After 20 min chromatin was isolated and immunoblotted for MCM3 or stained with Coomassie to show histones. (B) MCM3 chromatin association in (A) was quantified relative to control (mean ± SEM, n = 4). (C and D) Sperm nuclei were incubated in whole Xenopus egg extract supplemented with [α-32P]dATP. At either the time of DNA addition (C) or 20 min later (D), extract was additionally supplemented with 0, 25, 50, 100, 150, or 200 μM RL5a in DMSO. At the indicated times, the total amount of DNA synthesized was determined by trichloroacetic acid precipitation and scintillation counting. (E) Sperm nuclei were incubated in whole egg extract supplemented with DMSO or 200 μM RL5a. At the indicated times nuclear formation was assessed by either (i) phase contrast or (ii) UV microscopy. Scale bar, 20 μm. See also Figures S3 and S4.
	Figure 4. Effect of RL5a on Unlicensed and Pre-licensed DNA (A) Cartoon of the sequential loading of ORC, Cdc6, Cdt1, and MCM2–7 onto origin DNA. (B) Sperm nuclei were incubated in Xenopus egg extract. At the time of DNA addition (“unlicensed DNA”) or 15 min later (pre-licensed DNA), extract was optionally supplemented with geminin, DMSO, or the indicated concentrations of RL5a. After a further 20 min, chromatin was isolated and immunoblotted for ORC subunits (Orc1 and Orc2), Cdc6, and MCM3. The bottom of the gel was stained with Coomassie to show histones.
	Figure 5. Effect of RL5a on Licensing-Defective Extracts Sperm nuclei were incubated in Xenopus egg extract that had optionally been depleted of MCM3 or Cdc6 or which had been supplemented with geminin and which were further supplemented with 200 μM RL5a or DMSO. After 20 min, chromatin was isolated in 50, 100, or 200 mM KCl and then immunoblotted for ORC (Orc1 subunit) or Cdc6. (A and B) A representative series of blots is shown in (A). Blots from at least three separate experiments were quantified for the amount of chromatin-bound ORC (Orc1 subunit) and Cdc6. The mean signal ± SEM, relative to control chromatin isolated in 50 mM KCl, is plotted in (B). (C) Plasmid DNA (pET28) was incubated in a partially purified fraction of ORC supplemented with 2.5 mM ATP and either 25, 50, 100, 150, or 200 μM RL5a or DMSO. After 30 min, DNA was isolated in 100 mM KCl and then immunoblotted for Orc1 and Orc2. (D) Orc1 and Orc2 plasmid DNA association in (C) was quantified relative to recovered DNA and expressed in relation to control (mean ± SEM, n ≥ 5). See also Figure S5.
	Figure 6. ATP Requirement for ORC and Cdc6 DNA Binding (A) Whole Xenopus egg extract was desalted and then supplemented with 2.5 mM ATP or ATP-γ-S plus or minus geminin. Sperm nuclei were incubated in extract for 20 min and isolated in 50, 100, or 200 mM KCl, then immunoblotted for ORC (Orc1 and Orc2 subunits), Cdc6, Cdt1, and MCM3. The bottom of the gel was stained with Coomassie to show histones. (B) Sperm nuclei were incubated in whole Xenopus egg extract that had optionally been treated with geminin, apyrase, or ATP-γ-S. After 20 min, chromatin was isolated in 50, 100, or 200 mM KCl and then immunoblotted for ORC (Orc1 and Orc2 subunits) and MCM3. The bottom of the gel was stained with Coomassie to show histones. (C and D) Sperm nuclei were incubated in whole Xenopus egg extract that had optionally been treated with geminin, apyrase, ATP-γ-S, or RL5a. After 20 min, chromatin was isolated in 100 mM KCl and then immunoblotted for ORC (Orc1 and Orc2 subunits) and MCM3. The bottom of the gel was stained with Coomassie to show histones. (E and F) Xenopus egg extract was depleted of ATP by precipitation with polyethylene glycol, then supplemented with the indicated concentrations of ATP plus or minus 200 μM RL5a. Sperm nuclei were incubated in the reconstituted extract for 15 min, and the degree of licensing obtained was determined by assaying for replication in extract supplemented with geminin. Raw values for the mean ± SEM of three independent experiments (E) and a Lineweaver-Burk plot of the inverse values (F) are shown.
	Figure 7. Depiction of the Licensing Reaction and Its Inhibition by RL5a (A) U2OS and IMR90 cells were incubated in 1, 2, 5, 10, 20, 50, or 100 μM RL5a or DMSO for either 24, 48, 72, 96, or 120 hr and the relative cell number determined. Each data point presented is the mean ± SEM of three biological repeats each formed from three technical repeats. (B) (i) ORC initially binds weakly to DNA. (ii) In the presence of ATP or ATP-γ-S, ORC can bind tightly to DNA. This transition is inhibited by RL5a. (iii) Tightly bound ORC further recruits Cdc6 and Cdt1 to DNA. (iv) Double hexamers of MCM2–7 are clamped around DNA as the origin becomes licensed. This requires ATP hydrolysis.
	Supplementary Figure S1. Flow cytometry analysis of DNA content of cells at different stages in the screen (relates to Figure 1). Cells were taken through the steps of the screen as shown in Figure 1A. At different steps, cells were isolated and stained with DAPI and their DNA content analysed by flow cytometry. A. Cells after the first 16 hr thymidine block. B. Cells 12 hr after release from the first thymidine block at the point when the 2nd thymidine block was applied. C. Cells at the end of the 2nd(12 hr) thymidine block. D. Cells 6 hr after release from the 2nd thymidine block, at the point when RO3306 was added. E. Cells after 10 hr RO3306 treatment, at the point of test compound addition. F. Cells 8 hr after release from the RO3306 block, at the point when the final Mcm4 assay was typically performed.
	Supplementary Figure S3. SYPRO Ruby stained SDS-PAGE gels of chromatin isolated from Xenopus egg extract ± RL5a (relates to Figure 3). Chromatin was isolated from Xenopus egg extract, supplemented or not with RL5a, at the indicated times, over a sucrose cushion. Isolated chromatin was subjected to SDS-PAGE and gels were stained with SYPRO Ruby to visualize recovered proteins. A ‘no DNA’ control was included to facilitate identification of chromatin associated proteins. A. Egg extract was supplemented with 25, 50, 100, 150 and 200 µM RL5a and chromatin was isolated at 30 min; (i) 10% load, (ii) 90% load, light and dark exposures. B, C. Egg extract was supplemented with RL5a at either (B) 150 µM or (C) 200 µM, and chromatin was isolated at the indicated times. Light and dark exposures of 90% load are shown
	Supplementary Figure S4. Effect of RL5a after double-thymidine release (relates to Figure 3). Following release from a double-thymidine synchronisation, in the absence (A) or presence (B) of 10 µM RL5a, DNA content (PI) of U2OS cells was measured by flow cytometry at the indicated times: 0 hr (red), 4 hr (blue), 8 hr (orange) and 24 hr (green)
	Supplementary Figure S5. SYPRO Ruby stained SDS-PAGE gel of chromatin isolated from geminin treated Xenopus egg extract ± RL5a (relates to Figure 5). Chromatin was isolated from Xenopus egg extract, treated (or not) with geminin and supplemented (or not) with 25, 50, 100, 150 and 200 µM RL5a, over a sucrose cushion, at 20 min. Isolated chromatin was subjected to SDS-PAGE and the gel was stained with SYPRO Ruby to visualize recovered proteins. A ‘no DNA’ control was included to facilitate identification of chromatin associated proteins. Light and dark exposures are shown on the left and right respectively. The positions of Mcm2-7({}), Orc1 (•1), Orc2 (•2) and Orc3 (•3) are indicated.

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