Inactivation of Orc6 causes growth defects. (A) The Orc6 temperature-sensitive degron strain was constructed by transforming wild-type cells with a PCR product containing KanMX, CUP1 promoter, and degron cassette flanked with Orc6 homologous sequences. The integration of the degron cassette was confirmed by PCR. (B) Serial dilutions (1:5) of a wild-type control strain, orc6-td, mcm4-td, and an orc6-td strain with a plasmid carrying wild-type ORC6 were grown under the indicated conditions and examined after 2 d.

Orc6 is required for pre-RC maintenance. (A) Experimental outline. Wild-type and orc6-td cultures were grown to an OD₆₀₀ = 0.6 at room temperature in YP + Raffinose containing Cu²⁺ (time point 1) before adding α factor to arrest cells at G1 (time point 5). (Time point 6) Cells were switched into fresh YP + Galactose in the presence of α factor at 37°C to deplete Orc6-td. To assess whether cells could proceed to synthesize DNA, the cultures were released from α-factor arrest into fresh YP + Galactose at 37°C. (B) Orc6-td protein was depleted while other ORC subunits and Mcm2–7 remained. TCA-precipitated whole-protein samples from the indicated time points were assayed as described in Figure 2B. (C) The DNA content of samples from the indicated time points was analyzed by flow cytometry. Budding indices are shown to the right of each time point. (D) Depletion of Orc6 after pre-RC formation caused Mcm2–7 to dissociate from origins. Anti-Mcm2–7 ChIP samples and input from time point 6 in A were analyzed as described in Figure 2D. Chromosome XI is shown as an example (wild-type strain is blue, and orc6-td strain is orange).

Mapping of Orc6 protein-binding domains. (A) The C-terminal 62-amino-acid residues of Orc6 are sufficient for Orc1–5 binding. GST-Orc6 fusion proteins were bound to glutathione beads and washed before adding purified ORCΔ6. Total protein was assayed by silver staining after separation on SDS-PAGE. (B) Orc6 binds directly to Orc3 and Orc5. Individually expressed ORC subunits were incubated with bead-bound GST-Orc6 and were assayed by anti-Orc1, anti-Orc3, anti-Orc4, and anti-Orc5 Western analysis using antibodies SB13, SB3, SB6, and SB5, respectively. (C) Both the N- and C-terminal domains of Orc6 interact with Cdt1. GST-Orc6 fusion proteins were bound to glutathione beads and washed. (Top panel) Purified Cdt1 protein was incubated with GST-Orc6 and bound Cdt1 was assayed by immunoblotting. Ponceau S staining of glutathione bead-bound proteins is shown in the bottom panel.

ORC–CLO can only perform one round of Mcm2–7 loading. (A) Mcm2–7 complexes loaded by ORC–CLO are resistant to salt extraction (cf. lane 5). Pre-RCs were formed with ORC or ORC–CLO in ORC-depleted extract as for Figure 6B, and were incubated in buffer H/500mM NaCl for salt extraction. (B) ORC–CLO cannot load Mcm2–7 complexes reiteratively. Comparison between ORC–CLO and its Orc4-R267A-containing derivative ORC-4R–CLO showed they both load same amount of Mcm2–7 (cf. lanes 7,8).

Orc6 is required for pre-RC establishment. (A) Experimental outline. Wild-type and orc6-td cultures were grown to an OD₆₀₀ = 0.6 at room temperature in YP + Raffinose containing Cu²⁺ (time point 1) before adding nocodazole to arrest cells at G2/M (time point 2). (Time point 3) Galactose was added to 2% to induce UBR1 expression for 2 h. (Time point 4) Cells were then released into fresh YP + Galactose in the presence of α factor at 37°C to fully deplete the Orc6-td protein. To assess whether cells could proceed to synthesize DNA, the cultures were released from α-factor arrest into fresh YP + Galactose at 37°C. (B) Orc6-td protein was depleted while other ORC subunits and Mcm2–7 remained. TCA-precipitated whole-protein samples from indicated time points were separated by SDS–polyacrylamide gel electrophoresis, blotted, and probed with monoclonal anti-Mcm2–7 (AS1.1), anti-Orc6 (SB49), anti-Orc1 (SB13), anti-Orc3 (SB3), and anti-Orc4 (SB6) antibodies. (C) The DNA content of samples from the indicated time points was analyzed by flow cytometry. Budding indices are shown to the right of each time point. (D) orc6-td cells failed to load Mcm2–7 on origins. Pre-RC formation was detected by anti-Mcm2–7 ChIP hybridized on yeast 44K tiled DNA microarray. Log2 ratio of enrichment of anti-Mcm2–7 ChIP sample over input from time point 4 in A (establishment) was plotted as a function of its position along the chromosome. Chromosome XI is shown here as an example (wild-type strain is blue, and orc6-td strain is orange).

Orc6 is required for pre-RC assembly in vitro. (A) Pre-RC assembly assays with ORC and ORCΔ6 were performed with ORC-depleted G1 whole-cell extract (WCE) (ySC7) on 1039-base-pair (bp) ARS1-containing DNAs. Bead-associated proteins were analyzed by immunoblotting. Sequences of wild-type and mutant A and B2 elements of ARS1 are shown. (B) Orc6 is required for stable association of Cdt1 in the presence of ATPγS. Assembly reactions were performed with ATP or ATPγS on 1039-bp B2 + M2 ARS1-containing DNA.

Tethering Cdt1 to ORCΔ6 can recruit Mcm2–7 in vitro. (A) ORC–CLO forms a single complex. CLO was coexpressed with Orc1–5 using baculovirus infection of insect cells and purified by ion exchange chromatography. A Coomassie-stained, SDS–polyacrylamide gel showing the coelution of Orc1–5 and the CLO protein from the second column (Mono-Q, GE Healthcare) is shown. CLO associates stoichiometrically with Orc1–5. (B) ORC–CLO binds to B2 + M2 ARS1 DNA and loads Mcm2–7 efficiently in the presence of Cdc6. CLO was detected using anti-Cdt1 antiserum HM5352 (Randell et al. 2006). Note: In a typical pre-RC assembly reaction, ORC is ∼10-fold more abundant than Cdt1, even when the latter is stabilized by ATPγS. This was the reason for the strong CLO signals in B and C, and in Figure 7. (C) Mcm2–7 loading by ORC–CLO does not depend on endogenous Cdt1. Pre-RC assembly assays were performed with ORC- and Cdt1-depleted WCE.