The 9-1-1 DNA clamp subunit RAD1 forms specific interactions with clamp loader RAD17, revealing functional implications for binding-protein RHINO

The RAD9-RAD1-HUS1 complex (9-1-1) is a eukaryotic DNA clamp with a crucial role at checkpoints for DNA damage. The ring-like structure of 9-1-1 is opened for loading onto 50 recessed DNA by the clamp loader RAD17 RFC-like complex (RAD17-RLC), in which the RAD17 subunit is responsible for specificity to 9-1-1. Loading of 9-1-1 is required for activation of the ATR-CHK1 checkpoint pathway and the activation is stimulated by a 9-1-1 interacting protein, RHINO, which in-teracts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other inter-acting proteins may contain a RAD1-binding motif as well. Here, we show that vertebrate RAD17 proteins also have a putative RAD1-binding motif in their N-terminal regions, and we report the crystal structure of human 9-1-1 bound to a human RAD17 peptide incorporating the motif at 2.1 angstrom reso-lution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific in-teractions. Furthermore, we show that the RAD1-binding motif of RHINO disturbs the interaction of the N-terminal region of RAD17 with 9-1-1. Our results provide deeper understanding of how RAD17-RLC specifically recognizes 9-1-1 and imply that RHINO has a functional role in 9-1-1 loading/unloading and checkpoint activation.

Automated summary

The RAD9-RAD1-HUS1 complex (9-1-1) plays a crucial role in DNA damage checkpoints in eukaryotes. Loading of 9-1-1 onto recessed DNA is facilitated by the RAD17 RFC-like complex (RAD17-RLC) and is necessary for activation of the ATR-CHK1 checkpoint pathway. The interaction between 9-1-1 and the protein RHINO, which contains a RAD1-binding motif, is shown to disturb the interaction between RAD17 and 9-1-1. These findings provide a deeper understanding of how RAD17-RLC recognizes 9-1-1 and suggest a functional role for RHINO in 9-1-1 loading/unloading and checkpoint activation.