Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies...

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Eukaryotic DNA clamps, like PCNA and the 9-1-1 complex, are crucial for DNA transactions.
• The 9-1-1 complex, comprising RAD9, RAD1, and HUS1, activates the ATR-CHK1 pathway for DNA damage checkpoint control.
• The 9-1-1 complex features a ring structure and a RAD9 C-terminal tail (RAD9 C-tail), with regulatory roles.

Purpose of the Study:

• To biochemically and structurally analyze the intra- and inter-molecular interactions of the 9-1-1 complex.
• To elucidate the regulatory mechanisms of the RAD9 C-tail and the RHINO protein in checkpoint activation.

Main Methods:

• Biochemical assays to study protein-protein interactions.
• X-ray crystallography to determine the structure of the 9-1-1 complex with RHINO.
• Structural modeling of the quaternary 9-1-1-RHINO complex.

Main Results:

• The RAD9 C-tail binds to a hydrophobic pocket on the RAD9 subunit of the 9-1-1 ring.
• RHINO protein binds to the same hydrophobic pocket on RAD9, suggesting a shared interaction site.
• Crystal structure reveals RHINO's binding motif and informs a model of the 9-1-1-RHINO quaternary complex.

Conclusions:

• The RAD9 C-tail negatively regulates 9-1-1 DNA binding, while RHINO positively regulates checkpoint activation.
• The hydrophobic pocket on RAD9 is critical for both intramolecular (RAD9 C-tail) and intermolecular (RHINO) interactions.
• These findings provide insights into the dynamic regulation of the DNA damage checkpoint by the 9-1-1 complex.