TASP1-mediated cleavage of REV3L enhances the activity of DNA polymerase ζ in mammalian cells

DNA polymerase ζ (Polζ) is essential for replication across damaged DNA, yet the mechanisms governing its regulation in mammalian cells remain incompletely understood. The catalytic subunit REV3L is nearly twice the size of its yeast counterpart owing to a large vertebrate-specific insertion, and recent studies reported that REV3L undergoes proteolytic cleavage by the endopeptidase TASP1. Here, we show that REV3L cleavage generates two stable fragments, Nter-p70 and Cter-p300, which reassociate into a long-lived heterodimeric complex with enhanced polymerase activity. This interaction is stabilized by key residues within the catalytically inactive EXO domain. The cleaved Polζ complex associates with chromatin, binds nascent DNA, and undergoes ATR-dependent phosphorylation in response to replication stress. Importantly, preventing REV3L cleavage markedly impairs Polζ activity, reduces cisplatin-induced mutagenesis, and increases RAD51 nuclear foci and sister chromatid exchange, consistent with a compensatory shift toward homologous recombination–mediated repair. Together, our findings suggest that the vertebrate-specific expansion of REV3L necessitated cleavage to restore structural organization and optimize catalytic function, representing an evolutionary adaptation in DNA damage tolerance pathways. The TLS DNA polymerase ζ (Polζ) is essential for replication across damaged DNA. Here, the authors reveal that TASP1 mediated cleavage of REV3L, the catalytic subunit of Polζ, enhances Polζ activity. Blocking cleavage reduces mutagenesis and shifts repair toward homologous recombination showing the importance of proteolysis.