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.
	Figure2. 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 invitro 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 RL5ae.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.

Arias, Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. 2007, Pubmed

Arias, Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. 2007, Pubmed
Bell, ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. 1992, Pubmed
Blow, How dormant origins promote complete genome replication. 2011, Pubmed
Blow, Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system. 1987, Pubmed , Xenbase
Blow, A role for the nuclear envelope in controlling DNA replication within the cell cycle. 1988, Pubmed , Xenbase
Blow, Replication licensing and cancer--a fatal entanglement? 2008, Pubmed
Blow, Preventing re-replication of chromosomal DNA. 2005, Pubmed
Blow, Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. 1993, Pubmed , Xenbase
Blow, Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly. 1990, Pubmed , Xenbase
Blow, A model for DNA replication showing how dormant origins safeguard against replication fork failure. 2009, Pubmed
Bowers, ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication. 2004, Pubmed
Chang, Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication. 2015, Pubmed
Chong, Characterization of the Xenopus replication licensing system. 1997, Pubmed , Xenbase
Chong, Purification of an MCM-containing complex as a component of the DNA replication licensing system. 1995, Pubmed , Xenbase
Coster, Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. 2014, Pubmed
Cox, DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function. 1992, Pubmed , Xenbase
Evrin, A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. 2009, Pubmed , Xenbase
Evrin, In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization. 2013, Pubmed
Evrin, The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation. 2014, Pubmed
Feng, Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells. 2003, Pubmed
Ferenbach, Functional domains of the Xenopus replication licensing factor Cdt1. 2005, Pubmed , Xenbase
Frigola, ATPase-dependent quality control of DNA replication origin licensing. 2013, Pubmed
Gambus, MCM2-7 form double hexamers at licensed origins in Xenopus egg extract. 2011, Pubmed , Xenbase
Ge, Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories. 2010, Pubmed
Ge, Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. 2007, Pubmed
Gillespie, Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. 2001, Pubmed , Xenbase
Gillespie, Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins. 2012, Pubmed , Xenbase
Gillespie, Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts. 2004, Pubmed , Xenbase
Gillespie, Cell Cycle Synchronization in Xenopus Egg Extracts. 2016, Pubmed , Xenbase
Gillespie, Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication. 2000, Pubmed , Xenbase
Harvey, CpG methylation of DNA restricts prereplication complex assembly in Xenopus egg extracts. 2003, Pubmed , Xenbase
Hua, Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2. 1998, Pubmed , Xenbase
Ibarra, Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. 2008, Pubmed
Ilves, Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. 2010, Pubmed
Kang, Multiple functions for Mcm2-7 ATPase motifs during replication initiation. 2014, Pubmed
Klemm, Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. 1997, Pubmed
Lambert, Checkpoint responses to replication fork barriers. 2005, Pubmed
Li, The origin recognition complex: a biochemical and structural view. 2012, Pubmed
Lipford, Nucleosomes positioned by ORC facilitate the initiation of DNA replication. 2001, Pubmed
Liu, Replication licensing promotes cyclin D1 expression and G1 progression in untransformed human cells. 2009, Pubmed
Machida, Acute reduction of an origin recognition complex (ORC) subunit in human cells reveals a requirement of ORC for Cdk2 activation. 2005, Pubmed
McGarry, Geminin, an inhibitor of DNA replication, is degraded during mitosis. 1998, Pubmed , Xenbase
Montagnoli, Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells. 2004, Pubmed
Moreno, Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells. 2016, Pubmed
Moyer, Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. 2006, Pubmed
Nevis, Origin licensing and p53 status regulate Cdk2 activity during G(1). 2009, Pubmed
Newport, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. 1987, Pubmed , Xenbase
Oehlmann, The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation. 2004, Pubmed , Xenbase
Prokhorova, Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity. 2000, Pubmed , Xenbase
Randell, Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase. 2006, Pubmed
Remus, Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. 2009, Pubmed
Rowles, Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins. 1999, Pubmed , Xenbase
Rowles, Interaction between the origin recognition complex and the replication licensing system in Xenopus. 1996, Pubmed , Xenbase
Sasaki, Evidence for a mammalian late-G1 phase inhibitor of replication licensing distinct from geminin or Cdk activity. 2011, Pubmed , Xenbase
Seki, Stepwise assembly of initiation proteins at budding yeast replication origins in vitro. 2000, Pubmed
Sheehan, Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. 1988, Pubmed , Xenbase
Shreeram, Cell type-specific responses of human cells to inhibition of replication licensing. 2002, Pubmed
Sonneville, The dynamics of replication licensing in live Caenorhabditis elegans embryos. 2012, Pubmed
Speck, ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. 2005, Pubmed
Tada, Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. 2001, Pubmed , Xenbase
Teer, Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation. 2006, Pubmed
Tsakraklides, Dynamics of pre-replicative complex assembly. 2010, Pubmed
Vashee, Sequence-independent DNA binding and replication initiation by the human origin recognition complex. 2003, Pubmed , Xenbase
Wohlschlegel, Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. 2000, Pubmed , Xenbase
Woodward, Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress. 2006, Pubmed , Xenbase
Zwerschke, The Saccharomyces cerevisiae CDC6 gene is transcribed at late mitosis and encodes a ATP/GTPase controlling S phase initiation. 1994, Pubmed