The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure

  1. Hsiang-Chen Chou
  2. Kuhulika Bhalla
  3. Osama El Demerdesh
  4. Olaf Klingbeil
  5. Kaarina Hanington
  6. Sergey Aganezov
  7. Peter Andrews
  8. Habeeb Alsudani
  9. Kenneth Chang
  10. Christopher R Vakoc
  11. Michael C Schatz
  12. W Richard McCombie
  13. Bruce Stillman  Is a corresponding author
  1. Cold Spring Harbor Laboratory, United States
  2. John Hopkins University, United States
  3. Johns Hopkins University, United States

Abstract

The Origin Recognition Complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2 deficient cells were also large, with decompacted heterochromatin. Some ORC2 deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

Data availability

The data has been deposited to the Dryad database.

The following data sets were generated

Article and author information

Author details

  1. Hsiang-Chen Chou

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  2. Kuhulika Bhalla

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  3. Osama El Demerdesh

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  4. Olaf Klingbeil

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  5. Kaarina Hanington

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  6. Sergey Aganezov

    Department of Computer Science, John Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  7. Peter Andrews

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  8. Habeeb Alsudani

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  9. Kenneth Chang

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  10. Christopher R Vakoc

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1158-7180
  11. Michael C Schatz

    Department of Computer Science, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  12. W Richard McCombie

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
  13. Bruce Stillman

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    For correspondence
    stillman@cshl.edu
    Competing interests
    Bruce Stillman, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9453-4091

Funding

National Cancer Institute (P01-CA13106)

  • Bruce Stillman

National Cancer Institute (P50-CA045508)

  • Bruce Stillman

National Science Foundation (DBI-1627442)

  • Michael C Schatz

National Cancer Institute (P01-CA13106)

  • Christopher R Vakoc

National Cancer Institute (P01-CA13106)

  • W Richard McCombie

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Michael R Botchan, University of California, Berkeley, United States

Publication history

  1. Received: August 5, 2020
  2. Accepted: January 30, 2021
  3. Accepted Manuscript published: February 1, 2021 (version 1)
  4. Version of Record published: February 10, 2021 (version 2)

Copyright

© 2021, Chou et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Hsiang-Chen Chou
  2. Kuhulika Bhalla
  3. Osama El Demerdesh
  4. Olaf Klingbeil
  5. Kaarina Hanington
  6. Sergey Aganezov
  7. Peter Andrews
  8. Habeeb Alsudani
  9. Kenneth Chang
  10. Christopher R Vakoc
  11. Michael C Schatz
  12. W Richard McCombie
  13. Bruce Stillman
(2021)
The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure
eLife 10:e61797.
https://doi.org/10.7554/eLife.61797

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