The 9-1-1 DNA clamp subunit RAD1 forms specific interactions with clamp loader RAD17, revealing functional...

Area of Science:

• Molecular Biology
• Structural Biology
• Cellular Biology

Background:

• The RAD9-RAD1-HUS1 complex (9-1-1) functions as a eukaryotic DNA clamp essential for DNA damage checkpoints.
• The RAD17 RFC-like complex (RAD17-RLC) loads the 9-1-1 clamp onto DNA, with RAD17 conferring specificity.
• Activation of the ATR-CHK1 pathway relies on 9-1-1 loading, which is modulated by interacting proteins like RHINO.

Purpose of the Study:

• To investigate potential RAD1-binding motifs in other interacting proteins, specifically RAD17.
• To elucidate the structural basis of RAD17-RLC interaction with the 9-1-1 complex.
• To understand the role of RHINO in modulating RAD17-RLC-9-1-1 interactions.

Main Methods:

• X-ray crystallography was used to determine the structure of the human 9-1-1 complex bound to a RAD17 peptide.
• Biochemical assays were employed to analyze the interactions between RAD17, 9-1-1, and RHINO.

Main Results:

• The crystal structure revealed specific interactions between the N-terminal region of RAD17 and the RAD1 subunit of the 9-1-1 complex.
• A putative RAD1-binding motif was identified in the N-terminal region of vertebrate RAD17 proteins.
• The RAD1-binding motif of RHINO was shown to interfere with the binding of the RAD17 N-terminal region to the 9-1-1 complex.

Conclusions:

• The N-terminal region of RAD17 directly interacts with the RAD1 subunit of the 9-1-1 complex, explaining RAD17-RLC specificity.
• RHINO's interaction with 9-1-1 may regulate the loading or unloading of the 9-1-1 clamp, impacting checkpoint activation.
• These findings enhance the understanding of DNA damage checkpoint mechanisms involving the 9-1-1 clamp and its regulators.