Results: 5

1.
Fig. 2.

Fig. 2. From: The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.

The MBP-fused ORCs are active in binding to Cdc6 and DNA. Gel-shift assay of 32P-labeled ARS1 DNA with ORC, Cdc6, and MBP-fused ORC complexes or Orc1-5 subcomplex as indicated. The gel-shift assay was performed with 2 nM concentrations of the various ORCs, 0.2 nM of the concentrations labeled ARS1 DNA, and 0.8–2.4 nM of the concentrations Cdc6.

Zhiqiang Chen, et al. Proc Natl Acad Sci U S A. 2008 July 29;105(30):10326-10331.
2.
Fig. 5.

Fig. 5. From: The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.

The architecture of the yeast ORC. (A) A summary of ORC subunit interactions derived from the in vitro translation and IP experiments. The Orc6 subunit was not included in the analysis. (B) A summary of subunit mapping results. The approximate MBP position represents the location of the corresponding N or C terminus of the subunit onto which the MBP was fused. For an example, 1C refers to the average MBP position found in Orc(2-6)–Orc1–0MBP. (C) A proposed model for ORC interaction with Cdc6 and origin DNA. See Discussion for details.

Zhiqiang Chen, et al. Proc Natl Acad Sci U S A. 2008 July 29;105(30):10326-10331.
3.
Fig. 3.

Fig. 3. From: The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.

Detection of MBP in the 2D class averages of the MBP-fused ORC EM images. In each case, the upper row displays 10 class averages from the corresponding MBP-fused ORC and the lower row shows the same images at higher contrast level (contrast = 0.3). In the illustrations (Far Right), asterisks mark the observed MBP locations relative to ORC. The number of particles used for calculating each average is indicated. 1C refers to the ORC with MBP fused at the C terminus of Orc1 (ORC–Orc1–MBP). Likewise, the other complexes, 2C, 3N, 3C, 4N, 4C, and 5N also contain MBP at their N or C termini as indicated.

Zhiqiang Chen, et al. Proc Natl Acad Sci U S A. 2008 July 29;105(30):10326-10331.
4.
Fig. 4.

Fig. 4. From: The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.

In vitro subunit interaction. (A Upper) Genes encoding the individual ORC subunits, cloned into the transcription/translation vector (pCite-2a+), were transcribed and the resulting mRNA was translated individually and in various combinations, 5% of which was run on an SDS/PAGE gel as Input. The remainder of the translation mix was then immunoprecipitated with 20 μl Gamma Bind G Sepharose and washed three times and 50% of the IP mix was loaded onto SDS/PAGE gels. (A Lower and B–D) The IP of anti-Orc4 (A Lower), anti-Orc1 (B), anti-Orc2 (C), and anti-Orc3 (D) antibodies are shown. (A Lower Inset) A longer exposure of the gel, which was inserted over the top of the lighter image to observed Orc1 protein. The Orc6 subunit was not observed reliably in the IP lanes and hence we did not assign partners for this subunit.

Zhiqiang Chen, et al. Proc Natl Acad Sci U S A. 2008 July 29;105(30):10326-10331.
5.
Fig. 1.

Fig. 1. From: The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.

EM structures of the yeast ORC (Orc1-6) and Orc1-5 subcomplex. (A) A raw electron micrograph of the untagged ORC deeply stained by uranyl acetate showing various views. (Inset) Seven reprojections of the derived ORC 3D map (Left) and the corresponding 2D class averages (Right). (B) A comparison of three typical side views of ORC (Upper) with that of Orc1-5 (Lower) reveals the position of Orc6, as indicated by the red arrow. The number in each view refers to the raw particle images used for calculating the reference-free class average. The Greek letters α through η indicate the seven high-density regions in the averaged images. (C) Surface-rendered views of the 3D EM maps of ORC (Left) and Orc1-5 (Center). (Right) The difference map at the threshold of 3σ. The largest difference density represents the Orc6 position. b and c are views rotated 90° from a around a vertical and a horizontal axis, respectively.

Zhiqiang Chen, et al. Proc Natl Acad Sci U S A. 2008 July 29;105(30):10326-10331.

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