PT  - JOURNAL ARTICLE
AU  - Hsiang-Chen Chou
AU  - Kuhulika Bhalla
AU  - Osama El Demerdesh
AU  - Olaf Klingbeil
AU  - Kaarina Hanington
AU  - Sergey Aganezov
AU  - Peter Andrews
AU  - Habeeb Alsudani
AU  - Kenneth Chang
AU  - Christopher R. Vakoc
AU  - Michael C. Schatz
AU  - W. Richard McCombie
AU  - Bruce Stillman
TI  - The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure
AID  - 10.1101/2020.08.06.240358
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.08.06.240358
4099  - http://biorxiv.org/content/early/2020/08/06/2020.08.06.240358.short
4100  - http://biorxiv.org/content/early/2020/08/06/2020.08.06.240358.full
AB  - 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 StatementThe authors have declared no competing interest.