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. 2005 Dec 1;19(23):2827-36.
doi: 10.1101/gad.1369805.

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

David Y Takeda  1 , Yoshiyuki Shibata, Jeffrey D Parvin, Anindya Dutta
Affiliations

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

David Y Takeda et al. Genes Dev. .

Abstract

Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy.

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Figures

Figure 1.
Figure 1.
Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. (A) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10). After digestion with DpnI and NdeI (A) or NdeI alone (B), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2, pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. (C) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B. (D) C33a cells were transfected with the indicated plasmids and replication measured as in A. The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. (E) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.
Figure 2.
Figure 2.
Pre-RC components are loaded at the artificial origin. (A) Schematic of the plasmid pFR_Luc showing the two primer sets used in ChIP assays. One set is located proximal to the GAL4-binding sites and is denoted as “Origin” DNA, while the second set is located distal to the GAL4-binding sites and is denoted as “Non-origin” DNA. (B) ChIP using chromatin from HEK293 cells transfected with plasmids expressing GAL4-Orc2 (left panel) or Orc2 (right panel). The dark bars represent amplified Origin DNA, while the light bars represent amplified non-origin DNA. The inset shows the same plot at a different scale in order to show the results of the PCR reaction using anti-Orc2 and anti-Orc3. The Y-axis is the ratio of DNA in immunoprecipitate to that in input. (C) ChIP using chromatin from cells transfected with plasmids expressing GAL4-Cdc6 (left panel) or Cdc6 (right panel). The dark bars represent amplified Origin DNA, while the light bars represent amplified non-origin DNA. The Y-axis is the ratio of DNA in immunoprecipitate to that in input.
Figure 3.
Figure 3.
Replication requires Orc1 and Orc2 and is inhibited by Geminin overexpression. (A) HEK293A were cotransfected with a plasmid expressing GAL4-Cdc6 and siRNA oligos to GL2, Orc1, or Orc2. Plasmid replication was measured by the quantitating the relative amount of DpnI-resistant DNA as in Figure 1 (R/S) and values represented as the percentage of mock-transfected samples. (B) Immunoblotting of the samples in A with the indicated antibodies. (C) Following knock-down by siRNA of the indicated targets, cells were incubated in the presence of [3H]thymidine, and the amount of incorporated [3H]thymidine was determined by scintillation counting. Values were normalized to cell number as determined by MTT assay. (D) HEK293 cells were transfected with a plasmid expressing GAL4-Orc2 with increasing amounts of a second plasmid expressing wild-type Geminin. Replication was measured as in Figure 1. (E) HEK293 cells were transfected with plasmids expressing GAL4-Orc2 or GAL4-Cdc6 with increasing amount of Geminin expression plasmid as in D. The molar ratio of geminin-expressing plasmid to GAL4-Orc2 (or GAL4-Cdc6)-expressing plasmid is indicated. Extrachromosomal DNA was digested with DpnI + ExoIII or ExoIII alone, and replication was measured by quantitative real-time PCR (QPCR) using the primer set corresponding to origin DNA. The Y-axis is the DpnI-resistant DNA as measured by the ratio of QPCR signal from DNA digested with DpnI + ExoIII to DNA digested by ExoIII only. Identical results were obtained using the primer set that amplifies non-origin DNA (data not shown).
Figure 4.
Figure 4.
pFR_Luc replicates once per cell cycle with initiation localized to the GAL4-binding sites. (A) Schematic diagram of time course of experimental protocol. (B,C) HEK293 cells transfected with plasmids expressing GAL4-Orc2 (B) or GAL4-Cdc6 (C) and pFR_luc were released from a thymidine block into medium containing BrdU and harvested 0, 12, or 36 h later. Genomic and extrachromosomal DNA was isolated, digested, and loaded onto a cesium chloride gradient to separate DNA of different densities. Fractions were collected, and the amount of genomic DNA in each fraction was determined by measuring the absorbance at 260 nm (open circles); the amount of DpnI-resistant plasmid DNA was measured by quantitative PCR (filled squares). (D) Primer pairs used to measure the nascent-strand abundance assay on pFR_luc by real-time quantitative PCR. (E) Nascent-strand DNA was purified from HEK293 cells transfected with GAL4-Orc2 (dark bars) or GAL4-Cdc6 (light bars), and DNA was quantified by quantitative PCR. The results are normalized to the amount of non-origin DNA to allow comparisons between independent experiments. Data represent the average and standard deviation of two independent experiments performed in duplicate.
Figure 5.
Figure 5.
Activity of Cdc6 and Orc1 point mutants. (A) C33a cells were transfected with the indicated wild-type or mutant plasmid constructs of Cdc6 or Orc1, and replication of pFR_Luc was measured as in Figure 1. (WB) Walker B mutant; (S1) sensor-I mutant; (WHD) winged-helix-domain mutant. (B) Immunoblotting of samples in A and B using anti-GAL4. Asterisks indicate a nonspecific band used as a loading control. (C) Subcellular localization of different constructs as determined by immunofluorescence using antibodies to GAL4. The percent of cells showing nuclear, cytoplasmic, and nuclear + cytoplasmic location of the GAL4 fusion protein is indicated.
Figure 6.
Figure 6.
N terminus-deleted Orc1 and Orc2 can stimulate DNA replication. (A) C33a cells were transfected with wild-type or deletion constructs of Orc1 or Orc2, and replication of pFR_Luc was measured as in Figure 1. (B) Immunoblotting of samples in A using anti-Gal4 showing expression of the relevant GAL4 fusion proteins. The asterisk indicates a nonspecific band.
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