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The architecture of a eukaryotic replisome

Nature Structural & Molecular Biology volume 22, pages 976–982 (2015)Cite this article

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Abstract

At the eukaryotic DNA replication fork, it is widely believed that the Cdc45–Mcm2–7–GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol ɛ is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α–primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ɛ, first threading through the Mcm2–7 ring and then making a U-turn at the bottom and reaching Pol ɛ at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical replisome studies.

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Figure 1: Structure of the S. cerevisiae CMG helicase.
Figure 2: Structure of the S. cerevisiae CMGE leading-strand helicase-polymerase.
Figure 3: Rigid-body docking of CMG subunits into the CMGE density map with available crystal structures.
Figure 4: Subunit proximities within CMGE determined by chemical cross-linking with mass spectrometry readout (CX-MS).
Figure 5: Staged assembly of the eukaryotic replisome.
Figure 6: Architecture of the eukaryotic replisome.

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Acknowledgements

We would like to thank O. Yurieva and D. Zhang for the purification of CMG, Pol α, Pol ɛ and Ctf4. This work was funded by the US National Institutes of Health (GM103314 and GM109824 to B.T.C.; GM74985 and AG29979 to H.L.; and GM38839 to M.E.O'D.) and the Howard Hughes Medical Institute (M.E.O'D.).

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Authors and Affiliations

  1. Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA

    Jingchuan Sun, Zuanning Yuan & Huilin Li

  2. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA

    Jingchuan Sun, Zuanning Yuan & Huilin Li

  3. DNA Replication Laboratory, Rockefeller University, New York, New York, USA

    Yi Shi, Roxana E Georgescu, Brian T Chait & Michael E O'Donnell

  4. Howard Hughes Medical Institute, Rockefeller University, New York, New York, USA

    Roxana E Georgescu & Michael E O'Donnell

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  1. Jingchuan Sun
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Contributions

J.S., R.E.G., Y.S., B.T.C., H.L. and M.E.O'D. designed experiments. J.S., Y.S., R.E.G. and Z.Y. performed experiments. J.S., R.E.G., Y.S., Z.Y., B.T.C., H.L. and M.E.O'D. analyzed the data. H.L. and M.E.O'D. prepared the manuscript with input from all authors.

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Correspondence to Huilin Li or Michael E O'Donnell.

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Integrated supplementary information

Supplementary Figure 1 DNA binding and 3D EM reconstruction of the ScCMG complex.

(a) Determination of the Kd value of CMG to primed forked DNA by EMSA. Autoradiograph of CMG added to 1 nM 32P-primed fork DNA is to the left, and the quantitation is to the right. The dotted line represents the fit to a 1:1 binding event. (b) EMSA autoradiographs of primed 32P-forked (left) and unprimed forked (right). CMG and DNA were at concentrations of 100 nM DNA and 200 nM, respectively. For CMG-DNA EM grids, CMG was initially mixed at 592 nM and DNA was 1184 nM, after which samples were diluted to 57nM CMG prior to application to the grid. (c) Determination of the Kd value of CMGE to primed forked DNA by EMSA, similar to as described in panel (a). (d–g) 3D reconstruction of the ScCMG complex. (d) A selected region of a raw electron micrograph of purified yeast CMG in uranyl acetate stain. (e) A comparison of raw particle images (left column) with reference-free class averages (middle) and reprojections from the 3D map (right) in approximately corresponding views. (f) Gold-standard Fourier shell correlation and resolution estimation using the 0.143 criterion. (g) Euler angle distribution of all particles included in the final three-dimensional reconstruction. The size of each black dot relates to particle number.

Supplementary Figure 2 3D reconstruction of the CMGE complex.

(a) A selected region of a raw micrograph of the CMGE complexes. (b) Comparison of four raw CMGE particle images (top row) with their approximately corresponding class averages (middle row) and reprojections from the 3D map (bottom row). (c) Gold-standard Fourier shell correlation and resolution estimation using the 0.143 criterion. (d) Euler angle distribution of all particles included in the final three-dimensional reconstruction. The size of each black dot relates to particle number in the particular orientation. (e) 3D classification procedure used for reconstruction of the CMG-E 3D map. The first and forth models appeared to be broken complexes. The third model was only CMG, lacking any bound pol ε. These three models were discarded. The second model was apparently CMGE complex with fully occupied Pol ε. This model and the associated raw particles were selected for further refinement.

Supplementary Figure 3 Distribution of the Euclidean Cα-Cα distances of the peptide cross-links mapped onto the crystal structure of Pol2 (PDB http://www.pdb.org/pdb/search/structidSearch.do?structureId=4M8O).

The red bars represent the cross-link distances measured in the crystal structure. These distances are an approximation since Pol 2 is in the apo form in the CX-MS study, and the crystal structure is of the ternary complex.

Supplementary Figure 4 Intermolecular cross-link positions mapped onto the crystal structure of the catalytic polymerase Pol2.

The structure (4M8O) of the N-terminal catalytic polymerase of Pol ε. Balls show the positions of cross-links to Dpb2 (blue), Dpb3/4 (green), and Cdc45 (red).

Supplementary Figure 5 Selected views of different replisome complexes.

Left 5 panels: Comparison of replisome complexes visualized in near side views. Right 5 panels: Comparison of replisome complexes visualized in near top or slightly tilted top views. (a) Class averages of CMG complex. (b) Class averages of CMG-Ctf4. (c) Class averages of CMG-Ctf4-Pol α particles. Note the Pol α density marked by a red arrow in each panel is blurry, likely due to low occupancy and small contact area with the Ctf4 trimer. (d) Class averages of CMGE particles. (e) Class averages of CMGE-Ctf4 particles.

Supplementary Figure 6 Proposed possible DNA paths through the replisome.

(a) This diagram indicates a shorter DNA path if the leading strand exits the Mcms through the Mcm2/5 gate about half way through the Mcms. (b) This diagram indicates the nearby separate domain of the N-half active region of Pol2, if this region were to account for the lower observed density of Pol ε in CMGE. (c) This diagram explains the possibility that the CMG acts as a motor on the dsDNA leading strand product to push Pol ε, enabling Pol ε to serve as a strand displacing enzyme during leading strand fork progression.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 1752 kb)

3D EM map of CMGE

The video shows the relative positions of Pol epsilon and the N-terminal zinc fingers in the Mcm subunits within CMGE. (MP4 3376 kb)

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Sun, J., Shi, Y., Georgescu, R. et al. The architecture of a eukaryotic replisome. Nat Struct Mol Biol 22, 976–982 (2015). https://doi.org/10.1038/nsmb.3113

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