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

Hsiang-Chen Chou, View ORCID ProfileKuhulika Bhalla, Osama El Demerdesh, View ORCID ProfileOlaf Klingbeil, Kaarina Hanington, View ORCID ProfileSergey Aganezov, Peter Andrews, View ORCID ProfileHabeeb Alsudani, View ORCID ProfileKenneth Chang, View ORCID ProfileChristopher R. Vakoc, View ORCID ProfileMichael C. Schatz, View ORCID ProfileW. Richard McCombie, View ORCID ProfileBruce Stillman
doi: https://doi.org/10.1101/2020.08.06.240358
Hsiang-Chen Chou
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
2Graduate Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, NY
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Kuhulika Bhalla
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Osama El Demerdesh
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Olaf Klingbeil
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Kaarina Hanington
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Sergey Aganezov
3Department of Computer Science, Whiting School of Engineering, John Hopkins University, Baltimore, Maryland 21218
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Peter Andrews
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Habeeb Alsudani
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Kenneth Chang
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Christopher R. Vakoc
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Michael C. Schatz
3Department of Computer Science, Whiting School of Engineering, John Hopkins University, Baltimore, Maryland 21218
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W. Richard McCombie
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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Bruce Stillman
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11746
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  • For correspondence: stillman@cshl.edu
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Abstract

The Origin Recognition Complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre- RC complexes at origins of DNA replication. Here we report tiling-sgRNA CRISPR screens that show that each subunit of ORC and CDC6 are essential in human cells. Using an auxin-inducible degradation system, stable cell lines were created that ablate ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defect in the absence of ORC2 was 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.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted August 06, 2020.
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The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure
Hsiang-Chen Chou, Kuhulika Bhalla, Osama El Demerdesh, Olaf Klingbeil, Kaarina Hanington, Sergey Aganezov, Peter Andrews, Habeeb Alsudani, Kenneth Chang, Christopher R. Vakoc, Michael C. Schatz, W. Richard McCombie, Bruce Stillman
bioRxiv 2020.08.06.240358; doi: https://doi.org/10.1101/2020.08.06.240358
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The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure
Hsiang-Chen Chou, Kuhulika Bhalla, Osama El Demerdesh, Olaf Klingbeil, Kaarina Hanington, Sergey Aganezov, Peter Andrews, Habeeb Alsudani, Kenneth Chang, Christopher R. Vakoc, Michael C. Schatz, W. Richard McCombie, Bruce Stillman
bioRxiv 2020.08.06.240358; doi: https://doi.org/10.1101/2020.08.06.240358

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