RESEARCH ARTICLE Chk1 Activation Protects Rad9A from Degradation as Part of a Positive Feedback Loop during Checkpoint Signalling

Phosphorylation of Rad9A at S387 is critical for establishing a physical interaction with TopBP1, and to downstream activation of Chk1 for checkpoint activation. We have previ-ously demonstrated a phosphorylation of Rad9A that occurs at late time points in cells exposed to genotoxic agents, which is eliminated by either Rad9A overexpression, or con-version of S387 to a non-phosphorylatable analogue. Based on this, we hypothesized that this late Rad9A phosphorylation is part of a feedback loop regulating the checkpoint. Here, we show that Rad9A is hyperphosphorylated and accumulates in cells exposed to bleomy-cin. Following the removal of bleomycin, Rad9A is polyubiquitinated, and Rad9A protein lev-els drop, indicating an active degradation process for Rad9A. Chk1 inhibition by UCN-01 or siRNA reduces Rad9A levels in cells synchronized in S-phase or exposed to DNA damage, indicating that Chk1 activation is required for Rad9A stabilization in S-phase and during checkpoint activation. Together, these results demonstrate a positive feedback loop involv-ing Rad9A-dependend activation of Chk1, coupled with Chk1-dependent stabilization of Rad9A that is critical for checkpoint regulation