TTF2 promotes replisome eviction from stalled forks in mitosis

Area of Science:

• Cellular biology
• Molecular genetics
• DNA replication and repair

Background:

• Under-replicated DNA during mitosis compromises sister chromosome segregation, leading to genome instability.
• The E3 ubiquitin ligase TRAIP ubiquitylates the CMG helicase to disassemble stalled replisomes, cleave forks, and restore chromosome structure.

Purpose of the Study:

• To elucidate the molecular mechanisms by which TRAIP mediates replisome disassembly in mitosis.
• To investigate the roles of Cyclin B-CDK1 kinase and TTF2 in TRAIP-mediated replisome regulation.

Main Methods:

• Phosphorylation assays to determine TRAIP modification by Cyclin B-CDK1.
• Co-immunoprecipitation and biochemical assays to analyze protein-protein interactions (TRAIP, TTF2, CMG, DNA polymerase ε).
• Assessment of replisome disassembly, fork cleavage, and RNAPII eviction in cells with modified TRAIP and TTF2.

Main Results:

• TRAIP phosphorylation by Cyclin B-CDK1 is essential for replisome disassembly.
• TTF2 acts as a scaffold, tethering phosphorylated TRAIP to the CMG helicase and DNA polymerase ε via its N-terminal zinc finger and an adjacent peptide.
• This TRAIP-TTF2-pol ε complex facilitates CMG unloading and stalled fork breakage, while TTF2's ATPase activity is required for RNAPII eviction.

Conclusions:

• Mitotic replisome disassembly is a regulated process involving TRAIP phosphorylation and the scaffolding function of TTF2.
• A novel TRAIP-TTF2-pol ε bridge is critical for resolving under-replicated DNA and maintaining genome stability.
• TTF2 plays dual roles in mitosis, coordinating replisome disassembly and RNAPII eviction through distinct mechanisms.