The human origin recognition complex (ORC) is essential for pre-RC assembly, mitosis and maintenance of nuclear structure - Bhalla, Kuhulika; Demerdesh, Osama-El; Klingbeil, Olaf; Hanington, Kaarina; Aganezov, Sergey; Andrews, Peter; Alsudani, Habeeb; Chang, Kenneth; Vakoc, Christopher R.; Schatz, Michael C.; McCombie, Richard W.; Stillman, Bruce; et al. (Zenodo, 2021)
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.
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 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 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
Keywords: gpl-3.0-or-later; Origin Recognition Complex; tiling-sgRNA CRISPR screen; Initiation of DNA replication; Pre-replicative complex; CDC6; Nuclear Structure; copy number variation (CNV); SMASH analysis; ORC1; ORC2