Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis

Abstract

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

Keywords:

• Mitosis
• nucleolus
• Orc6
• phosphorylation
• replication

Acknowledgments

We thank members of the Prasanth laboratory, Dazhen Liu, Drs S. Baserga, and B. Stillman for providing reagents and insightful suggestions.

Author contributions

A.C., F.K., H.Z.O., M.L., M.K.A., R.P., D.C., Y-C.L., designed, performed, and analyzed most experiments. G.O., helped with statistics of the microscopy data; S.G.P. and K.V.P. supervised the project. F.K., H.Z.O., and S.G.P. wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

Data sharing not applicable – no new data generated.

Additional information

Funding

This work was supported by National Institutes of Health [R21AG065748; R01GM132458], and National Science Foundation [2243257; NSF science and technology center for quantitative cell biology] to KVP; and NSF [1818286, 2225264), NIH [GM125196] and CCIL awards to SGP.