The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure.

Hsiang-Chen Chou,Osama El Demerdesh,Olaf Klingbeil,Kaarina Hanington,Habeeb Alsudani,Sergey Aganezov,Christopher R Vakoc,Kenneth Chang,W Richard Mccombie,Bruce Stillman,Kuhulika Bhalla,Michael C Schatz,Peter Andrews

doi:10.7554/elife.61797

Hsiang-Chen Chou, Osama El Demerdesh + Show 11 more

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https://doi.org/10.7554/elife.61797

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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, 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.

Highlights

Cell division requires the entire genome to be duplicated once and only once during S-phase of the cell cycle, followed by segregation of the sister chromatids into two daughter cells
When we evaluate the distribution of log fold change (LFC) for all sgRNAs, we saw a negative correlation with annotated domains (AD) – that is sgRNAs targeting AD regions showed significantly higher depletion compared to those targeting non-annotated domain (NAD) regions (Figure 2—figure supplements 6a and 7a)
The ORC2-/- cell line believed to be a complete knockout via the use of 3 sgRNAs, one targeting the exon 4, and the others targeting the sixth and seventh introns retained a truncated form of ORC2 that could interact with ORC3 and was expressed from a mutated gene

Summary

Introduction

Cell division requires the entire genome to be duplicated once and only once during S-phase of the cell cycle, followed by segregation of the sister chromatids into two daughter cells. To ensure complete and correct duplication of genomes, the initiation of DNA replication is highly regulated and begins with the assembly of a pre-Replication Complex (pre-RC) at origins of DNA replication throughout the genome (Bell and Labib, 2016). Cdc binds to ORC, followed by the binding of Cdt1-Mcm to form head-to-head Mcm double hexamers to complete the formation of the pre-RC (Araki, 2011; Bell and Labib, 2016; Bleichert et al, 2017; Evrin et al, 2009; Heller et al, 2011; Remus et al, 2009). During S phase, cyclin-dependent protein kinase (CDK) and the Cdc7-Dbf4-dependent protein kinase

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