Energetically equivalent structural transitions in the Rad17-Rad9-Hus1-Rad1-Rhino complex underlie the sequential...

Activation of the ATR-dependent DNA damage response (ATR-DDR) is well characterized; however, the molecular mechanisms underlying its maintenance and inactivation remain largely elusive. Rhino is the least understood component of ATR-DDR. Structural modeling and binding free energy calculations revealed structural remodeling involving Rad17, Rad9-Hus1-Rad1 (9-1-1), and Rhino during ATR-DDR progression. Biochemical and computational analyses revealed the competitive binding of Rad17 and Rhino to the 9-1-1 complex, suggesting a structural transition from the Rad17-9-1-1 complex to the Rhino-9-1-1 complex. The presence of two conserved KYxxL+ motifs in Rhino suggests that it bridges the two 9-1-1 complexes. This enables the polymerization of multiple 9-1-1 complexes through Rhino and explains the long-standing discrepancy between the conventional model and experimental observations of Rad17 and Rad9 foci. Furthermore, structural analysis of the Rad9 C-terminal tail revealed its ability to compete with both Rhino and Rad17, leading to disassembly of the checkpoint complex and providing a mechanism for checkpoint inactivation. Quantum chemical calculations revealed comparable binding free energies for intermediate complexes. These observations suggest that the Rad17-9-1-1-Rhino complex undergoes energetically equivalent structural transitions, providing a mechanistic basis for the sequential progression of ATR-DDR.