# Energetically equivalent structural transitions in the Rad17–Rad9–Hus1–Rad1–Rhino complex underlie the sequential progression from activation through maintenance to inactivation of the ATR-dependent DNA damage response

## TL;DR

The study reveals how structural changes in a DNA damage response complex help regulate the activation, maintenance, and inactivation of the ATR-dependent DNA damage response.

## Contribution

The study identifies energetically equivalent structural transitions in the Rad17–Rad9–Hus1–Rad1–Rhino complex that underlie the progression of the ATR-dependent DNA damage response.

## Key findings

- Rhino bridges two 9–1–1 complexes through conserved KYxxL+ motifs, enabling polymerization.
- Rad9's C-terminal tail competes with Rhino and Rad17, leading to checkpoint inactivation.
- Binding free energies of intermediate complexes are comparable, supporting energetically equivalent transitions.

## Abstract

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.

Graphical Abstract