Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

Abstract

Replication of eukaryotic chromosomes occurs once every cell division cycle in normal cells and is a tightly controlled process that ensures complete genome duplication. The origin recognition complex (ORC) plays a key role during the initiation of DNA replication. In human cells, the level of Orc1, the largest subunit of ORC, is regulated during the cell division cycle, and thus ORC is a dynamic complex. Upon S phase entry, Orc1 is ubiquitinated and targeted for destruction, with subsequent dissociation of ORC from chromosomes. Time lapse and live cell images of human cells expressing fluorescently tagged Orc1 show that Orc1 re-localizes to condensing chromatin during early mitosis and then displays different nuclear localization patterns at different times during G1 phase, remaining associated with late replicating regions of the genome in late G1 phase. The initial binding of Orc1 to mitotic chromosomes requires C-terminal amino acid sequences that are similar to mitotic chromosome-binding sequences in the transcriptional pioneer protein FOXA1. Depletion of Orc1 causes concomitant loss of the mini-chromosome maintenance (Mcm2-7) helicase proteins on chromatin. The data suggest that Orc1 acts as a nucleating center for ORC assembly and then pre-replication complex assembly by binding to mitotic chromosomes, followed by gradual removal from chromatin during the G1 phase.

Keywords: Cell Cycle; DNA Replication; DNA-binding Protein; FOXA1; Mitosis; Origin Recognition Complex; Protein-DNA Interaction.

FIGURE 1.

Characterization of a monoclonal antibody targeting human Orc1. A, Coomassie Brilliant Blue-stained gel showing step for purification of the Orc1-ΔN400 fragment from E. coli cells. B, epitope mapping for the Orc1 antigen. The fragments of MBP-fused Orc1 were subjected to immunoprecipitation (IP) using the Orc1 78-1-172 monoclonal antibody, and then Western blots were performed with anti-MBP antibody. Only the full-length (FL) MBP-Orc1 and MBP-Orc1-ΔN400 versions but not other N-terminal truncated mutants were immunoprecipitated. C, silver-stained gel showing that increasing amounts of beads cross-linked to Orc1 78-1-172 monoclonal antibody, but not IgG, can immunoprecipitate endogenous Orc1 protein as well as the exogenously expressed GFP-Orc1 fusion protein from HeLa cells expressing GFP-Orc1. D, immunoblot detecting Orc1 and GFP in immunoprecipitates similar to those shown in C. E, Orc1 78-1-172 monoclonal antibody can co-immunoprecipitate other subunits of ORC, including Orc3 and Orc4 as detected by immunoblot.

FIGURE 2.

Cell cycle dynamics of ORC assembly. A, flow cytometry profile of DNA content of double thymidine blocked and released HeLa suspension cells. Cell cycle profile of asynchronous and synchronized cells at the indicated hours after release are shown; x axis shows propidium iodide-area levels, and y axis shows cell count at different time points after release. B, expression levels of pre-RC proteins and cell cycle markers in synchronized HeLa nuclear extracts were studied by Western blot. Ponceau S-stained blots are shown as loading controls. C, immunoprecipitation of Orc1, Orc2, Orc3, or control IgG from double thymidine block and release synchronized HeLa S3 nuclear extracts. Co-immunoprecipitation of Orc1, Orc2, Orc3, and Orc4 were investigated by Western blots as indicated.

FIGURE 3.

Orc1 associates with mitotic chromosomes. Tetracycline-inducible GFP-Orc1 U2OS cell line was followed by time-lapse live cell imaging through mitosis. Highlighted by an arrow is a cell followed through mitosis (panels a–x in A and panels a–u in B) at indicated time points (min means minutes, and h means hours). Orc1 is loaded onto chromatin during prophase and stays on chromatin throughout mitosis, after telophase Orc1 becomes diffusely distributed in daughter nuclei (panels v–x). Scale bar denotes 5 μm. A and B represent images from two separate videos.

FIGURE 4.

Orc1 domains associate with mitotic chromosomes. A, domain structures of human FOXA1 and Orc1 proteins. Sequence alignment of FOXA1 DBD and Orc1 is shown. TAD, transactivation domain; WH, winged helix domain; A, Walker A motif; B, Walker B motif; I, motif I; II, motif II. Hs, Homo sapiens (human); Pan troglodytes (chimpanzee); Cf, Canis familiaris (dog); Bt, Bos taurus (bovine); Mm, Mus musculus (mouse); Rt, Rattus norvegicus (rat); Md, Monodelphis domestica (opossum) (100). In Orc1, sequences mean Orc1 protein sequences have 100 amino acid intervening residues. It suggests that Orc1 contains two motifs separated by 100 amino acid residues. Blue residues represent identity between FOXA1 and Orc1, and red residues represent similarity. B, localization of GFP-Orc1 or different GFP-NLS-Orc1 fragments in mitotic U2OS cells as indicated. U2OS cells were transiently transfected, and live cell images were captured. Each of the constructs in panels b–f have an NLS present. C, localization of GFP-NLS-Orc1(555–575) (motif I) during mitosis using live cell imaging of transfected U2OS cells. D, localization of GFP-NLS in methanol-fixed U2OS cells. Following transfections are with a plasmid expressing the protein: E same as D, except GFP-NLS-Orc1(672–740) (motif II). F same as D except GFP-Orc1(1–861).

FIGURE 5.

Orc1 shows extensive mitotic chromosome association before Orc2, and depletion of Orc1 causes loss of Mcm3 from chromatin. A, YFP-Orc1 localization relative to Orc2 localization in U2OS cells. In late mitosis YFP-Orc1 appears on chromatin in the daughter nuclei prior to Orc2, which remains in the cytoplasm (panels a–a″), and then Orc2 localizes to nuclei after telophase (panels b–b″). Orc2 was detected using the pAb205 antibody. Scale bar equals 5 μm. B, prominent MCM3 staining (red) on chromatin in control (luciferase) siRNA-treated cells (panel a) as compared with loss of MCM3 staining in Orc1 siRNA-treated U2OS cells using siRNA targeting the coding region (panel b) or the 3′UTR of endogenous Orc1 (panel c). DNA was stained with DAPI. Scale bar represents 5 μm. C, labeling of MCM3 on chromatin in control (luciferase siRNA)-treated U2OS cells stably expressing YFP-Orc1 (panels a and a′). YFP-Orc1 cell line treated with Orc1 siRNA targeting the coding region of endogenous and exogenous Orc1 shows loss of MCM3 from chromatin (panels b and b′). The arrowhead denotes a cell showing partial knockdown of Orc1 (as seen by YFP-Orc1) showing low levels of MCM3 loading. YFP-Orc1 cell line treated with Orc1 siRNA targeting the 3′UTR of endogenous Orc1 (panels c and c′). Note the rescue of MCM3 loading onto chromatin showing that siRNA-resistant exogenous YFP-Orc1 can functionally complement endogenous Orc1.

FIGURE 6.

Orc1 shows differential patterning in human cells. A, GFP-Orc1 localization in HeLa cells. Cells were transiently transfected with GFP-Orc1, and localization pattern was investigated in live HeLa cells. Image stacks were collected and deconvolved. Scale bar denotes 7 μm. B, visualization of YFP-Orc1 in living cells (MCF7 cells). Image stacks of cells expressing YFP-Orc1 were collected and deconvolved (from supplemental movie 3). Scale bar is equal to 10 μm. Note the change in the Orc1 pattern, initially appearing as uniformly distributed punctate spots (panels a–c) and later relocalizing to specific large foci. Images were captured every 20 min for ∼15 h.

FIGURE 7.

Spatial patterning of Orc1 occurs during G₁ phase. Asynchronously growing MCF7 cells expressing YFP-Orc1 were fixed and immunostained for MCM3 (orange, a and b) or PCNA (red, a′, and b′) or visualized for YFP-Orc1 (green, a″ and b″). Nuclei were detected by DAPI straining (blue, a‴, and b‴). Orc1 is present only in G₁ cells (MCM3-positive and PCNA-negative). Scale bar denotes 5 μm.

FIGURE 8.

Orc1 localization in G₁ phase overlaps with an inherited spatiotemporal pattern of DNA replication. A, scheme for the experiment. Human U2OS cells expressing YFP-Orc1 (green) were transfected for 15 min with dUTP Alexa 594 (red) (see “Experimental Procedures”). Following washing out the label and incubating for ∼12–14 h (ensuring the cells progress from S phase in generation 1 to G₁ of generation 2), live cells were imaged every 5 min using a Delta Vision optical sectioning deconvolution instrument (Applied Precision) on a Olympus microscope with a ×63 1.4NA objective. B, top two panels represent significant overlap of YFP-Orc1 with the dUTP Alexa 594, both in nuclear and nucleolar periphery (merge is panels a and b). YFP-Orc1 and dUTP Alexa 594 do not show any overlap but are immediately adjacent to each other (panels c–c″), and (panels d–d″) show the expected most common situation of no overlap between YFP-Orc1 and dUTP Alexa 594. Scale bar denotes 5 μm. Overlap was seen in 6% of all YFP-Orc1-positive cells. C, cells expressing YFP-Orc1 were incubated for 10 min with BrdU, following which BrdU was extensively washed out. Following ∼12–14 h incubation at 37 °C (ensuring the cells enter from S phase in generation 1 to G₁ of generation 2), cells were fixed in 2% formaldehyde. BrdU was detected using anti-BrdU mouse mAb (red) in YFP-Orc1 (green)-positive cells. Orc1 was observed both in the nuclear and nucleolar periphery. In a population of cells Orc1 showed significant overlap with BrdU-positive regions. Scale bar denotes 5 μm.