Final checkpoint for resolving unfinished DNA replication sites during cell division identified

The molecular mechanism underlying the final step in processing DNA replication sites that remain unfinished during mitosis (cell division) has been identified.

According to a study published on the 26th in the international journal Science, the transcription termination factor TTF2 acts as a bridge, while TRAIP tags unfinished DNA replication forks for emergency stalling and disassembly.

Eukaryotic cells must replicate their chromosomes and segregate them accurately during each cell cycle. However, in species with large genomes, some late-replicating regions risk remaining unreplicated when mitosis begins.

When animal cells enter mitosis and cyclin-dependent kinase 1 (CDK1) is activated, the TRAIP ubiquitin ligase triggers disassembly of the chromosome replication machinery. TRAIP promotes ubiquitination of the CDC45-MCM-GINS (CMG) helicase, and the tagged CMG is removed by the p97 ATPase, causing the replisome to collapse.

Until now, however, little was known about the molecular mechanism that regulates TRAIP during mitosis and processes unfinished DNA regions.

According to the researchers, CDK1 initiates ubiquitination of the CMG helicase. CDK1 phosphorylates a site on TRAIP that is essential for CMG disassembly during mitosis.

Phosphorylated TRAIP is recognized by the TTF2 ATPase. TTF2 is known to remove RNA polymerase II from mitotic chromosomes.

However, TTF2’s ATPase activity was not essential for CMG helicase disassembly. Instead, disassembly was mediated by two elements at the TTF2 N terminus: a pair of zinc fingers that bind phosphorylated TRAIP and a short motif that binds DNA polymerase epsilon.

Through this mechanism, TTF2 connects TRAIP to the replisome at mitotic onset, promoting CMG helicase ubiquitination and disassembly and activating a mitotic DNA repair pathway associated with sister chromatid exchange.

The researchers said, “TRAIP-dependent CMG ubiquitination acts on a single DNA replication fork, causing cleavage and rapid repair,” adding that “this process can occur only during mitosis because it depends on mitotic CDK1 activity and the cytoplasmic TTF2 ATPase.”

They added, “Because fork cleavage is restricted to the DNA strand originally encircled by the CMG helicase, it produces two sister chromatids that can be separated later, avoiding the harmful consequences of random cleavage.”