. 2010 Oct 8;40(1):99-111.
A WD-repeat Protein Stabilizes ORC Binding to Chromatin
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A WD-repeat Protein Stabilizes ORC Binding to Chromatin
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Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.
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Figure 1. ORCA Is a Highly Conserved ORC-Binding Protein
(A) Schematic representation of ORCA domain structure. Simple Modular Architecture Research Tool (SMART)-based domain predictions reveal three leucine-rich repeats at the N terminus and five WD repeats at the C terminus. (B) Sequence alignment shows high conservation of ORCA among higher eukaryotes (see the Supplemental Experimental Procedures for accession numbers). The top block represents alignment of LRR-3 (aa 90–113 human) and the lower block is WD40 repeat-1 (aa 390–430 human). Note that on the basis of bioinformatics prediction, only a conserved WD40-containing protein was found as a possible ortholog in
Drosophila. (C) ORCA associates with ORC. Immunoprecipitation with T7 mAb in cells transfected with T7-ORCA followed by immunoblots with antibodies against T7, Orc1, Orc2, and MCM2 reveals specific interaction of T7-ORCA with Orc1 and Orc2. Mouse IgG (mIgG) was used as the control. (D) Reverse IP with Orc2 mAb shows Orc2 can efficiently pull down endogenous ORCA from human cells. (E) ORCA interacts with multiple components of preRC. IP with ORCA antibody shows the interaction with ORC subunits, Cdt1, and Geminin. See also Figure S1.
Figure 2. ORCA Exists in Multiple Subcomplexes in the Cell
(A) Glycerol gradient sedimentation analysis of ORCA complex on material immunoprecipitated with ORCA antibodies. The corresponding molecular weight markers are labeled. Note that ORCA, Orc2, and Orc3 cosediment in fractions 12–14. (B) Association of ORCA with ORC in human cells. HeLa nuclear extract fractionated over a Superdex 200 gel filtration column and fractions analyzed for ORCA and various ORC subunits by immunoblot. Molecular weight markers are labeled on top of the panel. Note the multiple peaks of ORCA corroborating its existence in multiple subcomplexes.
Figure 3. ORCA Association with Chromatin Is Cell-Cycle Regulated
(A) Immunostaining of human cells with ORCA-specific antibody shows differential staining of ORCA, with some cells showing strong staining, while others lacking it. (B) YFP-ORCA-expressing MCF7 cells were pre-extracted and immunostained with MCM3 and PCNA. Note that the YFP-ORCA-expressing cells show punctate labeling of MCM3 and lack of PCNA, pointing to the presence of ORCA protein in G1 cell population. DNA is counterstained with DAPI (blue). Scale bars in (A) and (B) represent 10 µm. (C) Statistical analysis of MCM/PCNA profile in YFP-ORCA-expressing MCF7 cells. Note that ORCA is predominantly present in cells that show MCM+/PCNA– (60.3% ± 6.1%), representing G1 cells. ORCA-expressing, MCM+/PCNA+ cells denoting cells in S phase is 11.8% ± 8.2%, and MCM–/PCNA– is 27.9% ± 8.1%. The graph represents mean values plus standard deviation from four independent experiments (n = ~100 cells in each experiment). (D) Levels of ORCA during the cell cycle. ORCA immunoblot of whole-cell extract from cells synchronized during specific stages of the cell cycle. Note the increase in ORCA during G1 phase. (E) Biochemical fractionation of ORCA. Chromatin fractionation from cells synchronized during different stages of the cell cycle show accumulation of ORCA in G1 cells (S3 fraction) and decrease during G1/S and S. “*” denotes the cross-reacting band of ORCA pAb. Note that the cross-reacting band appears in the cytosolic fraction and is therefore not found in any of the experiments where nuclear extracts were used (Figures 1 and 2). MEK2 is shown as a control for chromatin fractionation. Flow cytometry profiles are shown on top of the panel. S2 represents cytosolic fraction, S3 nuclear soluble and MNase released nuclear fraction, and P3 the insoluble and MNase resistant nuclear fraction. Bar diagrams in (Db) and (Eb) represent the relative abundance of ORCA throughout cell cycle (level in G1 is being considered 100%). See also Figure S2 and Movie S1.
Figure 4. ORCA Colocalizes with ORC at Heterochromatic Structures
(A) IF with Orc2 pAb and HP1α antibodies after pre-extraction procedure in YFP-ORCA-expressing MCF7 cells reveals colocalization of ORCA with Orc2 and HP1α at heterochromatic regions. (B) Triple immunolabeling with antibodies against ORCA, telomere binding protein Trf2, and centromere protein AnaC shows colocalization of ORCA with centromeric and pericentromeric heterochromatin in MCF7 cells. (C) Immunostaining of Orc2 and HP1α in YFP-ORCA-expressing stable U2OS cells shows the complete overlap of ORCA and Orc2. (D) Immunostaining of Trf2 and AnaC in YFP-ORCA-expressing stable U2OS cells shows colocalization of YFP-ORCA at telomeres but not at centromeres. (E and F) Similar overlap of endogenous ORCA and YFP-Orc1 in U2OS cells at telomeres (E) but not at centromeres (F). The scale bar represents 10 µm. See also Figure S3.
Figure 5. WD40 Repeat of ORCA Mediates ORC and Chromatin Association
(A) Schematic representation of various truncation mutants of ORCA. Each of the mutants contains an epitope tag—T7 or YFP—at their N terminus for IP or IF analyses, respectively. (B) IP in cells expressing various T7-ORCA mutants with T7 antibody and analysis of Orc2 by immunoblot. Note that only ORCA constructs possessing the WD40 domain efficiently interact with Orc2 (T7-ORCA.128–647; 270–647; 1–647). (C) YFP-tagged truncated ORCA constructs and their chromatin binding as detected by detergent pre-extraction procedure followed by formaldehyde fixation. Note that only the constructs having WD repeat domain associate with chromatin. DNA was stained with DAPI (blue). The scale bar represents 10 µm. (D) Immunoblot analysis of YFP-tagged truncated ORCA constructs shows association of FL (1–647) and WD containing fragments (128–647 and 270–647) with chromatin (P). YFP-ORCA mutant constructs lacking WD (1–127 and 1–270) appear in nonchromatin-associated detergent-extracted supernatant (S) fraction. (E) YFP-ORCA.270–647 localizes to heterochromatin as evident by colocalization with HP1α and Orc2. The scale bar represents 10 µm. See also Figure S4.
Figure 6. ORCA Tethers ORC to Chromatin
(A) Schematic representation of the 2-6-3 CLTon locus in human U2OS cells (adapted and modified from Janicki et al. ). The chromatin locus is visualized by Cherry-LacI staining. (B) Cells were cotransfected with YFP-LacI and CFP-Orc2 or with YFP-LacI-ORCA and CFP-Orc2. YFP-LacI does not recruit CFP-Orc2 to the locus, whereas YFP-LacI-ORCA can efficiently recruit CFP-Orc2 to the chromatin locus. (C) Cells cotransfected with YFP-LacI-ORCA.1-270 or YFP-LacI-ORCA.270-647 and CFP-Orc2 show that only YFP-LacI-ORCA.270-647 recruits Orc2 to the locus. (D) Cells transfected with YFP-LacI-ORCA and not YFP-LacI alone show accumulation of MCM3 to the locus, demonstrating that ORC binding to this locus is functional. Scale bars represent 10 µm. See also Figure S5.
Figure 7. ORCA Is Required for Loading ORC to Chromatin
(A) Immunoblot analysis after siRNA depletion of ORCA with two independent siRNA oligonucleotides shows >90% depletion in U2OS cells. “*” denotes the cross-reacting band. Tubulin was used as the loading control. (B) Chromatin fractionation in ORCA depleted U2OS cells and immunoblot analysis. Note that in the ORCA-siRNA treated cells, there is significant reduction of Orc2 from P3 fraction and a concomitant accumulation in S2. Similar reduction is apparent for Orc1, Orc3, and Orc4. MEK2 was used as a control for chromatin fractionation. SF2/ASF, a splicing factor, is used as loading control. (C) Immunoblot analysis after depletion of ORCA in human primary diploid fibroblasts WI38. Note the increase in levels of p21 and Cyclin E and decrease in Cyclin B upon ORCA depletion. (D and E) Cell-cycle profile by flow cytometry of WI38 cells and hESCs depleted of ORCA shows increased accumulation in G1 phase. (F and G) Percentage of PCNA positive cells (by immunofluorescence) in control and ORCA-depleted WI38 and hESCs. The graphs represent mean values plus standard deviation from three independent experiments (n = ~400). (H) Chromatin fractionation in ORCA depleted WI38 cells and immunoblot analysis. Note the significant reduction of Orc2 and 3 and MCM2,3, and 5 on chromatin (P) and the increase in p21 in ORCA-depleted cells. “%” on the right side of (B) and (H) denotes the amount of protein left on chromatin after ORCA depletion. See also Figure S6.
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Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Adaptor Proteins, Signal Transducing / genetics
Adaptor Proteins, Signal Transducing / metabolism*
Chromatin Assembly and Disassembly*
Fluorescent Antibody Technique
Heterochromatin / metabolism*
Origin Recognition Complex / genetics
Origin Recognition Complex / metabolism*
Protein Interaction Domains and Motifs
Recombinant Fusion Proteins / metabolism
Adaptor Proteins, Signal Transducing
Origin Recognition Complex
Recombinant Fusion Proteins
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