Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• The human Origin Recognition Complex (ORC) initiates DNA replication and influences chromatin organization, centrosome biology, and cytokinesis.
• Orc6, the smallest ORC subunit, is poorly conserved and recent studies suggest its role in S-phase progression and DNA damage response via ATR-dependent phosphorylation at T229.

Purpose of the Study:

• To investigate the role of Orc6 phosphorylation at T195 during mitosis.
• To determine the impact of Orc6 phosphorylation on ORC function, S-phase progression, and cellular localization.

Main Methods:

• Site-directed mutagenesis to create phosphomimetic Orc6 mutants (T195E).
• Cell cycle analysis and western blotting to assess Orc6 phosphorylation and protein interactions.
• Immunofluorescence microscopy to determine Orc6 localization within the cell.

Main Results:

• CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis.
• The phosphomimetic T195E Orc6 mutant impedes S-phase progression.
• Phosphorylated Orc6 exhibits more robust association with ORC, particularly outside of G1, suggesting a role in preventing replication licensing.
• Orc6 and phosphorylated Orc6 localize to nucleolar organizing centers and regulate ribosome biogenesis.

Conclusions:

• Phosphorylation of Orc6 at T195 during mitosis plays a critical role in preventing DNA replication.
• Orc6 phosphorylation influences ORC complex stability and localization, impacting cell cycle control.
• Orc6's role extends to regulating ribosome biogenesis, highlighting its multifaceted functions.