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

J Biol Chem. 2023 Apr;299(4):103061. doi: 10.1016/j.jbc.2023.103061. Epub 2023 Feb 24.

Abstract

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 5' 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 interacts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other interacting 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 Å resolution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific interactions. 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.

Keywords: 9-1-1; DNA clamp; RAD17; RHINO; checkpoint; clamp loader; crystal structure; protein-protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carrier Proteins* / metabolism
  • Cell Cycle Proteins* / metabolism
  • DNA / metabolism
  • DNA Damage
  • DNA-Binding Proteins / metabolism
  • Exonucleases* / metabolism
  • Humans

Substances

  • Carrier Proteins
  • Cell Cycle Proteins
  • DNA
  • DNA-Binding Proteins
  • Exonucleases
  • Rad1 protein, human
  • Rad17 protein, human