ABSTRACT
The six subunit ORC is essential for initiation of DNA replication in eukaryotes. Cancer cell-lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2 or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵* Co-first-authors
The title, abstract, and discussion have been revised to be more concise and focused. Figure 2C has been updated to clarify that ORC2 was completely knocked out. The previous data were obtained from whole-cell lysates extracted from whole mouse liver, whereas the updated data are from isolated hepatocytes, providing a more purified cell population. Figure 3C has been removed to avoid potential confusion for readers. Figure 4B has been revised for greater concise. Figure 6 has been separated into two parts (Figures 6 and 7), with the addition of a western blot in Fig 6D to demonstrate the deletion of ORC1 and ORC2 in isolated hepatocytes.
