Article | Published:

Crystal structure of the eukaryotic origin recognition complex

Nature volume 519, pages 321326 (19 March 2015) | Download Citation

Abstract

Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1–5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2–7 (MCM2–7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the RSCB Protein Data Bank under accession code 4XGC.

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Acknowledgements

We thank the beamline scientists at beamlines 8.3.1 of the Advanced Light Source (Berkeley Lawrence National Laboratory), 23-ID-B of the Advanced Photon Source (Argonne National Laboratory) and X25 of the National Synchrotron Light Source (Brookhaven National Laboratory) for technical support with X-ray diffraction data collection, and the Nogales laboratory (University of California, Berkeley) for EM support. We also thank A. Fisher and Y. Li for assistance with insect cell cultures. This work was supported by the National Institutes of Health (GM071747 to J.M.B. and CA R37-30490 to M.R.B.) and by a fellowship from the UC Berkeley Miller Institute for Basic Research in Science (to F.B.).

Author information

Affiliations

  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA

    • Franziska Bleichert
    •  & James M. Berger
  2. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94720, USA

    • Michael R. Botchan

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Contributions

F.B. performed all biochemical and crystallization experiments, collected X-ray diffraction data and determined the structure with guidance from J.M.B. All authors interpreted and discussed results, and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Michael R. Botchan or James M. Berger.

Extended data

Supplementary information

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  1. 1.

    Supplementary Information

    DESCRIPTION

Videos

  1. 1.

    Structure of Drosophila ORC

    In the video, ORC starts off in the autoinhibited state as observed in crystals. ORC is first shown in the cartoon representation with individual subunits colored differentially (Orc1 – orange, Orc2 – green, Orc3 – blue, Orc4 – purple, Orc5 – gold, Orc6 – deepsalmon). Subsequently, the molecular surface is shown with the WH domains in light tints of their respective AAA+ domain color to illustrate domain swapping, which is best visualized in the side views with the Orc1 AAA+ and Orc2 WH domains removed. Remaining in the side view, the Orc1 AAA+ domain is shown next, which then morphs between autoinhibited (as observed in the crystal) and prospective remodeled (“activated”) conformations. The remodeled structure is then shown in cartoon format, with the ISMs and β-hairpin wings of ORC subunits rendered as surface in red and cyan, respectively. A close-up view of the central channel is next provided, illustrating the shallow spiral formed by the ISMs and β-hairpin wings. Finally, a 360° view of ORC in the remodeled conformation is shown in cartoon representation.

  2. 2.

    Comparison of Drosophila ORC crystal and EM structures

    The ORC crystal structure (Orc1 – orange, Orc2 – green, Orc3 – blue, Orc4 – purple, Orc5 – gold, Orc6 – deepsalmon) is docked into the ATPγS-bound Drosophila ORC EM volume (gray surface, EMD-247914). In the second part of the video, Orc1 is morphed into a remodeled (“activated”) conformation. The close agreement between the EM map and the ORC crystal structure, but not the remodeled ORC conformation, suggests that the autoinhibited state of ORC as observed in crystals corresponds to the predominant species in solution.

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https://doi.org/10.1038/nature14239

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