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

Chou, Hsiang-Chen, Bhalla, Kuhulika, Demerdesh, Osama El, Klingbeil, Olaf, Hanington, Kaarina, Aganezov, Sergey, Andrews, Peter, Alsudani, Habeeb, Chang, Kenneth, Vakoc, Christopher R, Schatz, Michael C, McCombie, W Richard, Stillman, Bruce (February 2021) The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure. eLife, 10. ISSN 2050-084X

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.

Item Type:	Paper
Subjects:	bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics Investigative techniques and equipment organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > cell cycle organs, tissues, organelles, cell types and functions > cell types and functions > cell functions organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line organs, tissues, organelles, cell types and functions > cell types and functions > cell types organs, tissues, organelles, cell types and functions > cell types and functions > cell types organs, tissues, organelles, cell types and functions > cell types and functions > cell types organs, tissues, organelles, cell types and functions > cell types and functions Investigative techniques and equipment > CRISPR-Cas9 organs, tissues, organelles, cell types and functions > organelles, types and functions > mitosis organs, tissues, organelles, cell types and functions > organelles, types and functions organs, tissues, organelles, cell types and functions
CSHL Authors:	Vakoc, Christopher R. Stillman, Bruce W.
Communities:	CSHL labs > Stillman lab CSHL labs > Vakoc lab CSHL Cancer Center Program CSHL Cancer Center Program > Cancer Genetics and Genomics Program CSHL Cancer Center Program > Gene Regulation and Inheritance Program
SWORD Depositor:	CSHL Elements
Highlight:	Stillman, Bruce W.
Depositing User:	CSHL Elements
Date:	1 February 2021
Date Deposited:	11 Feb 2021 18:11
Last Modified:	09 Feb 2024 21:17
PMCID:	PMC7877914
URI:	https://repository.cshl.edu/id/eprint/39896

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