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Hossain_Supplemental Figure 1 GFP-PACT GFP-PACT Motif I GFP-PACT Motif II A. MG132 (1µM) GFP Tubulin GFP-PACT Pericentrin GFP-PACT GFP-PACT Pericentrin Fig. S1. Expression and localization of Orc1 PACT in U2OS cells. Transient transfection of GFP-Orc1-PACT into U2OS cells. (A) U2OS cells were transiently transfected with GFP-Orc1 PACT, GFP-Orc1-PACT motif I and GFP-Orc1PACT motif II for 16 hours and further incubated for 8 hours in the presence or absence of 1 µm MG132, a proteasome inhibitor. The expression levels of Orc1 PACT proteins were determined by immunoblotting with GFP antibody. Equal loading of proteins in different lanes was confirmed by tubulin immunoblot. (B) Transiently overexpressed GFP-Orc1PACT protein in U2OS cells was co-stained with (for centrosome) and pericentrin (pericentriolar matrix) antibodies. Lower panel is at higher magnification.

A CyclinA Cdk2 H1a CyclinA Cdk2*H1a Orc1 K i Orc1 Hossain_Supplemental Figure 2 Kʹ i CyclinA Cdk2 H1a P P Orc1^CyclinA Cdk2 H1a P Orc1^CyclinA Cdk2*H1a B CyclinE Cdk2 H1a CyclinE Cdk2*H1a Orc1 CyclinE Cdk2 H1a P K i P Orc1^CyclinE Cdk2 Fig. S2. Kinetic models for Orc1 inhibition of Cyclin-CDK kinase activities on Histone H1 substrate. (A) The inhibition of cyclin A-Cdk2 kinase activity on histone H1a phosphorylation with Orc1 behaving as a mixed, non-competitive inhibitor. (B) The inhibition of cyclin E-Cdk2 kinase activity on histone H1a phosphorylation with Orc1 behaving as a competitive inhibitor only.

Hossain_Supplemental Figure 3 A. 66 45 MW (kda) Cyclin A-CDK2 66 45 MW (kda) 2 116 3 3 Pull Down (2%) Input (5%) -Orc1 A-A -Orc1 R15Q Cyclin E-CDK2 MW (kda) -Orc1 A-A -Orc1 R15Q 66 45 WB: Cyclin A WB: Cyclin E Fig. S3. Human Orc1 Interaction with Cyclin E-CDK2 or Cyclin A-CDK2 kinase. (A) A Coomassie Brilliant Blue stained gel of purified Cyclin A-CDK2 and Cyclin E- CDK2 kinase purified from baculovirus expression system (left and middle panel). Right panel shows purified -Orc1 wild type protein as well as its mutant derivatives (F89S, R15Q and E127G) purified from bacteria. MW stands for protein molecular weight marker in kilodalton. (B) In vitro interaction between Orc1 and the kinases in a pull down assay. The resin bound -Orc1 wild type or its mutant derivatives were incubated with purified Cyclin E-CDK2 or Cyclin A-CDK2. The specific binding of the kinases was detected by western blot using antibodies against Cyclin A or Cyclin E. Recombinant protein served as control protein in the assay.

Hossain_Supplemental Figure 4 A. C. GFP-CID-PACT GFP-CID PACT GFP-CID PACT Pericentrin Centrin 2 CEP17 SAS-6 GFP-CID-PACT GFP-CID-PACT Pericentrin GFP-CID PACT GFP-CID PACT Centrin 2 Centrin 2 GFP-CID PACT GFP-CID PACT CEP17 SAS-6 CEP17 SAS-6 Fig. S4. Localization of GFP-Orc1.CID. PACT in U2OS cells. Overexpression of GFP-Orc1-CID-PACT in U2OS cells. (A) U2OS cells were transiently transfected with GFP-Orc1-CID-PACT and the overexpressed protein was counter stained with (for centrosome) and pericentrin (pericentriolar matrix) antibodies. Lower panel is at higher magnification. (B) Transiently overexpressed GFP-Orc1-CID-PACT protein in U2OS cells were co-stained with (for centrosome) and Centrin-2 (centriole) antibodies. Lower panel is at higher magnification. (C) The GFP-Orc1-CIDPACT transfected U2OS cells was co-stained with CEP17 (grandmother centriole marker) and SAS-6 (daughter centrioles or pro-centrioles marker). Lower panel is at higher magnification.

Hossain_Supplemental Figure 5.25.5.75 1..25.5.75 1. (µm) Cyclin A/Cdk2 Cyclin E/Cdk2 - Orc1-CID - Orc1-CID-PACT Fig. S5. Inhibition of Cyclin A-CDK2 or cyclin E-CDK2 kinase. The inhibition of cyclin E-Cdk2 or cyclin A-Cdk2 kinase activity on histone H1a phosphorylation with different molar amounts (,.25,.5,.75 and 1 µm) of - Orc1-CID (1-25 aa Orc1) or -Orc1-CID-PACT. Concentrations of Cyclin-CDK2 and histone H1a in the assay were1 nm and.5 µm, respectively.

A. 2 INPUT (2 %) -Orc1 R15Q -Orc1 W88A Streptavidin beads - - - Pull Down(2%) -Orc1 R15Q -Orc1 W88A -Orc1 R15Q -Orc1 W88A - - - Hossain_Supplemental Figure 6 Biotinylated H4K2me2 Peptide (1µg) 116 66 45 2 -Orc1 Bound (nm) 16 12 8 4 Orc1 WT Orc1 F89S Orc1 R15Q Orc1 E127G Orc1 W88A 3 6 9 12 15 -Orc1 Input (nm) Fig. S6. -Orc1 binding to H4K2me2 peptide. (A) Biotinylated H4K2me2 peptide (1µg) was bound to streptavidin beads and incubated with 1µg -Orc1 wild type or its mutant protein. (B) Different concentration of -Orc1 wild type or its mutants were titrated for binding with 1µg of H4K2me2 peptide bound to streptavidin beads. Background subtraction was done with -Orc1 proteins bound to the beads only. Bands were quantified as described in methods section.

Hossain_Supplemental Fig. 7 Tetracycline (µg/ml).4882813.65625.1953125.39625.78125.15625.3125.625.125.25.5 1. GFP-Orc1 WT 116 * ** WB: Orc1 WB: Tubulin GFP-Orc1 R15Q 116 * ** WB: Orc1 WB: Tubulin Fig. S7. Controlled expression of the transgene from stable cell lines. Western blot of whole cell extracts of U2OS Flp-in stable cell lines expressing either GFPOrc1 WT wild type or GFP-Orc1 R15Q mutant protein. The transgenes were induced for 3 days (72hours) with the indicated amounts of tetracycline. Monoclonal antibody against Human Orc1 was used to detect endogenous as well as GFP-Orc1 proteins. -Tubulin served as a control for equal loading of each sample. Single and double asterisk marks indicates the GFP-Orc1 transgene and endogenous Orc1, respectively.

A. 3 Control GFP-Orc1 WT GFP-Orc1 R15Q Hossain_Supplemental Figure 8 No. of Cells (x1 5 ) 25 2 15 1 5 1 2 3 4 5 Days Fold Increase 6 5 4 3 2 1 Control GFP-Orc1 WT GFP-Orc1 R15Q 1 2 3 4 5 Days Fig. S8. Cell proliferation of GFP-Orc1 stable cell lines. U2OS Flp-in stable cell lines expressing either GFPOrc1 WT wild type or GFP-Orc1 R15Q mutant protein as well as control U2OS TreX cells were induced with tetracycline (.5 µg/ml) from day. Each of the cell lines were seeded in triplicate at a density of 1x1 5 cells a day before tetracycline addition. The cells were counted using Countess Automated Cell Counter (Invitrogen). The total number of cells for each cell line was followed for 5 days (A) and fold increase in cell number compared to day (B). The experiment was done in triplicate with standard deviations.

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