The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure
- Cold Spring Harbor Laboratory, United States
- John Hopkins University, United States
- 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.
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Data from: The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure.Dryad Digital Repository, doi:10.5061/dryad.59zw3r25f.
Article and author information
Author details
Christopher R Vakoc
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
- Michael R Botchan, University of California, Berkeley, United States
Version history
- Received: August 5, 2020
- Accepted: January 30, 2021
- Accepted Manuscript published: February 1, 2021 (version 1)
- 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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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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