Dysregulation of DNA polymerase κ recruitment to replication forks results in genomic instability

Translesion synthesis polymerases (TLS Pols) are required to tolerate DNA lesions that would otherwise cause replication arrest and cell death. Aberrant expression of these specialized Pols may be responsible for increased mutagenesis and loss of genome integrity in human cancers. The molecular events that control the usage of TLS Pols in non‐pathological conditions remain largely unknown. Here, we show that aberrant recruitment of TLS Polκ to replication forks results in genomic instability and can be mediated through the loss of the deubiquitinase USP1. Moreover, artificial tethering of Polκ to proliferating cell nuclear antigen (PCNA) circumvents the need for its ubiquitin‐binding domain in the promotion of genomic instability. Finally, we show that the loss of USP1 leads to a dramatic reduction of replication fork speed in a Polκ‐dependent manner. We propose a mechanism whereby reversible ubiquitination of PCNA can prevent spurious TLS Pol recruitment and regulate replication fork speed to ensure the maintenance of genome integrity.