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Published February 25, 2021 | Version v1
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The human origin recognition complex (ORC) is essential for pre-RC assembly, mitosis and maintenance of nuclear structure

Authors/Creators

  • 1. Cold Spring Harbor Laboratory
  • 2. Johns Hopkins University

Description

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.

Notes

  1. Tiling_sgRNA_CRISPR_screen.tar.gz
    • ATCACG.fastq.gz    initial time point for HCT116 cell line Replicate1
    • TTAGGC.fastq.gz    initial time point for HCT116 cell line Replicate2
    • TGACCA.fastq.gz    final time point for HCT116 cell line Replicate1
    • GGCTAC.fastq.gz    final time point for HCT116 cell line Replicate2
    • CTTGTA.fastq.gz    initial time point for RPE1 cell line
    • GAGTGG.fastq.gz    final time point for RPE2 cell line
    • HCT116_rep1.csv - LFC values
    • HCT116_rep2.csv - LFC values
    • RPE1.csv - LFC values
    • ReadMe.txt file 
       
  2. SMASH.zip
    • HCT116p53WT folder
      • HCT116p53WT_S6_R1_001.fastq.gz 
      • HCT116p53WT_S6_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • HCT116p53null folder
      • HCT116p53null_S2_R1_001.fastq.gz
      • HCT116p53null_S2_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • ORC1null folder
      • ORC1null_S3_R1_001.fastq.gz
      • ORC1null_S3_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • ORC2null folder
      • ORC2null_S4_R1_001.fastq.gz
      • ORC2null_S4_R2_001.fastq.gz
      • All other file with extensions .ps, .pdf, .txt are outputs of analysis using SMASH
    • ReadMe.txt
       
  3. ORC2null_ONT.zip
    • Alignments Folder - Alignment files
      • ORC2null.sort.bam.bai
      • ORC2null.sort.bam
    • SV_inference Folder - 
      • ORC2_CRISPR_sniffles.vcf - Analysis with Sniffles v1.0.11
      • ORC2_CRISPR_pbsv.vcf - Analysis with PBSV v2.2.0
    • ReadMe.txt

Funding provided by: National Cancer Institute
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000054
Award Number: P01-CA13106

Funding provided by: Cancer Center Support Grant*
Crossref Funder Registry ID:
Award Number: P50-CA045508

Funding provided by: Cancer Center Support Grant
Crossref Funder Registry ID:
Award Number: P50-CA045508

Files

HCT116p53null_1000000_bins_Rout.txt

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Additional details

Related works

Is cited by
10.7554/eLife.61797 (DOI)
Is source of
10.5061/dryad.59zw3r25f (DOI)