Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, Illinois, USA.
University Laboratory High School, Urbana, Illinois, USA.
Mol Cell Biol. 2024;44(7):289-301. doi: 10.1080/10985549.2024.2356880. Epub 2024 Jun 12.
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.
人类起始识别复合物（ORC）不仅需要启动 DNA 复制，还涉及多种细胞功能，包括染色质组织、中心体生物学和胞质分裂。ORC 的最小亚基 Orc6 在真核生物中保守性较差。我们实验室的最新研究表明，人类 Orc6 不需要复制许可，但需要 S 期进展。此外，ATR 依赖性磷酸化 Orc6 的 T229 位点与 S 期的 DNA 损伤反应有关。在这项研究中，我们证明 CDK 依赖性磷酸化 Orc6 的 T195 位点发生在有丝分裂期间。虽然 T195 位点的磷酸化似乎不是有丝分裂退出所必需的，但表达 Orc6 的磷酸模拟 T195E 突变体的细胞会阻碍 S 期的进展。此外，磷酸化形式的 Orc6 与 ORC 更强烈地结合，并且 Orc6 与 ORC 结合的时间点也在 G1 期之外，这支持了 Orc6 可能防止 Orc1-5 在 G1 期之外发挥许可作用的观点。最后，Orc6 和磷酸化的 Orc6 定位于核仁组织中心，并调节核糖体生物发生。我们的研究结果表明，T195 位点磷酸化的 Orc6 可以阻止复制。
