Chou, Hsiang-Chen (July 2021) The role of human ORC2 in DNA replication, mitosis and organization of the nucleus. PhD thesis, Cold Spring Harbor Laboratory.
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Abstract
In eukaryotic cells, the entire genome is duplicated only once each cell division cycle during S-phase and is followed by segregation of the sister chromatids to the two daughter cells. A pre-Replication Complex (pre-RC) is assembled at every origin prior to replication initiation and is assembled in a stepwise manner. It begins with the hexameric Origin Recognition Complex (ORC) 1-6 subunits binding to replication origins. Unlike yeast Saccharomyces cerevisiae ORC that remains a stable complex throughout the entire cell division cycle, in human cells the ORC complex dissociates soon after the pre-RC is activated at the beginning of S phase and the ORC1 subunit is degraded. Reports in the literature suggest that ORC1 and ORC2 in human cells are not entirely essential, however, CRISPR/Cas9 screens with guide RNAs that target the entire open reading frame of ORC1-6, CDC6, CDT1 and MCM2-7 showed that these proteins are all essential to cell survival. Indeed, the CRISPR/Cas9 screening points to essential domains in each of these proteins. Previous studies showed depletion of ORC2 or ORC3 in human cells using siRNA caused defects in S-phase and also during mitosis, however, depletion of these proteins using iv siRNA is a slow process. Here I used the auxin regulated mAID degron system to construct cell lines in which ORC2 or ORC3 were removed from cells and the resulting cell cycle phenotypes investigated. First, human cells were transduced with LTR-mAID-ORC2, which is resistant to a specific CRISPR-mediated single guide RNA (sgRNA) via retrovirus transduction into cell lines expressing the OsTIR1 protein, a ubiquitin ligase from rice that promotes degradation of proteins linked to an auxin inducible domain (mAID). Then the endogenous ORC2 gene was mutated using a CRISPR/Cas9 with a sgRNA that inactivates the gene. 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. This abnormal cell division phenotype then persists through mitosis causing abnormal chromosome segregation including the presence of lagging chromosomes, micronuclei and eventually apoptosis. The nuclei of ORC2 deficient cells were also large, with decompacted heterochromatin. 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 DNA replication, chromosomes segregation and nuclear organization.
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