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

Chou, H. C., Bhalla, K., El Demerdash, O., Klingbeil, O., Hanington, Kaarina, Aganezov, S., Andrews, P., Alsudani, H., Chang, K., Vakoc, C. R., Schatz, M. C., McCombie, W. R., Stillman, B. (August 2020) The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure. BioRxiv. (Unpublished)

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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.

Item Type: Paper
Subjects: organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > cell proliferation
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > origin recognition complex
CSHL Authors:
Communities: CSHL labs > Chang lab
CSHL labs > McCombie lab
CSHL labs > Schatz lab
CSHL labs > Stillman lab
CSHL labs > Vakoc lab
Depositing User: Sasha Luks-Morgan
Date: 6 August 2020
Date Deposited: 08 Jun 2021 17:39
Last Modified: 29 Apr 2024 15:25
URI: https://repository.cshl.edu/id/eprint/40205

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