TopBP1 assembles nuclear condensates to switch on ATR signaling

Area of Science:

• Cellular biology
• Molecular biology
• Biochemistry

Background:

• The ATR (Ataxia Telangiectasia and Rad3-related) checkpoint pathway is vital for maintaining genomic stability during DNA replication stress.
• TopBP1 is a key activator of ATR, but its precise mechanism of action remains incompletely understood.

Purpose of the Study:

• To investigate the self-assembly properties of TopBP1 and its role in ATR activation.
• To elucidate the molecular mechanisms underlying ATR signaling amplification.

Main Methods:

• Utilized an optogenetic platform to induce and observe TopBP1 self-assembly.
• Characterized the physical properties and dynamics of TopBP1 condensates.
• Investigated the functional consequences of TopBP1 condensation on ATR/Chk1 signaling.
• Performed in vitro liquid-liquid phase separation assays with purified TopBP1.

Main Results:

• TopBP1 self-assembles into functional, micrometer-sized condensates composed of nanoparticle clusters.
• These condensates are dynamic, reversible, and co-localize with ATR pathway proteins.
• TopBP1 condensation acts as a molecular switch, amplifying ATR activity to phosphorylate Chk1 and slow replication forks.
• Disruption of key residues in TopBP1's intrinsically disordered domain impairs condensation and ATR/Chk1 signaling.
• Purified TopBP1 undergoes liquid-liquid phase separation in vitro under physiological conditions.

Conclusions:

• TopBP1 condensation is a critical, regulated mechanism for amplifying ATR signaling.
• The assembly of TopBP1 condensates, driven by multivalent interactions, acts as the molecular switch for ATR pathway activation.
• This study reveals a novel mechanism for controlling DNA replication checkpoint signaling.