RAD9A Stabilization and Destabilization Processes in Checkpoint Maintenance and Checkpoint Recovery
Osorio Zambrano, William Frend
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The phosphorylation of the sensor-checkpoint Rad9A is essential for Chk1 activation in S-phase and during the G2/M checkpoints. Previous studies have demonstrated a late phosphorylation of Rad9A after DNA damage. Rad9A late phosphorylation requires the previous phosphorylation of S387, and it is not detected when Rad9A is overexpressed. Since Rad9A and TopBP1 interact through a phosphorylated S387 for ATR activation, it has been hypothesized that the late, damage-dependent phosphorylation on Rad9A is part of a positive feedback loop involved in checkpoint maintenance. This thesis shows that Rad9A is hyperphosphorylated and accumulates in cells exposed to DNA damage, and that this prevents Rad9A polyubiquitination and degradation. This work also shows that Rad9A polyubiquitination and degradation is observed in cells recovering from DNA damage, and that there are active processes regulating Rad9A degradation. This research further shows that Chk1 is required for the stabilization and accumulation of Rad9A after DNA damage, and that there is a positive feedback loop involving Rad9A and Chk1. Rad9A acetylation is observed in conjunction with polyubiquitination, and Rad9A deacetylation following DNA damage prevents Rad9A polyubiquitination and degradation. This work also shows evidence of Brca1 and Rad9A association at the time of Rad9A polyubiquitination and degradation suggesting a Brca1 involvement in this process. In summary, this thesis shows a new positive feedback loop between Rad9A and Chk1 for checkpoint maintenance and the stabilization of Rad9A through its deacetylation following DNA damage to prevent Rad9A polyubiquitination and degradation.