Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol

The bypass of DNA lesions by translesion synthesis (TLS) polymerases is a critical step for DNA damage tolerance, allowing the completion of DNA synthesis. It has been under debate whether TLS-mediated bypass restarts stalled forks or occurs post-replicationally. We developed cell imaging techniques based on proximity ligation to monitor the recruitment of TLS polymerases Polκ and Polη to DNA adducts. We show that this recruitment is adduct specific, with Polκ being preferentially recruited to benzo[a]pyrene diol epoxide (BPDE) lesions and Polη to cisplatin lesions. The recruitment depends on the primase-polymerase PrimPol, which reprimes downstream of stalled forks to restart DNA synthesis. TLS polymerase deficiency results in the accumulation of single-stranded DNA (ssDNA) gaps in an adduct-specific manner, which are processed into double-strand breaks (DSBs). Our findings argue that TLS occurs mainly behind the restarted replication fork in order to fill PrimPol-derived gaps and is essential to suppress the nucleolytic conversion of ssDNA gaps into cytotoxic DSBs in a lesion-specific manner.