Catalytic and non-catalytic functions of DNA polymerase kappa in translesion DNA synthesis

Translesion DNA synthesis (TLS) is an essential process that allows cells to bypass lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase kappa (Polκ) has the unique ability to bypass minor groove DNA adducts in vitro. However, Polκ is also required for cells to overcome major groove DNA adducts but the basis of this requirement is unclear. Here, we combine CRISPR base editor screening technology in human cells with TLS analysis of defined DNA lesions in Xenopus egg extracts to unravel the functions and regulations of Polκ during lesion bypass. Strikingly, we show that Polκ has two main functions during TLS, which are differentially regulated via Rev1 binding. On the one hand, Polκ is essential to replicate across minor groove DNA lesions in a process that depends on PCNA ubiquitylation but is independent of Rev1. On the other hand, via its cooperative interaction with Rev1 and ubiquitylated PCNA, Polκ stabilizes the Rev1-Polζ extension complex on DNA to allow extension past major groove DNA lesions and abasic sites, in a process that is independent of Polκ catalytic activity. Together, our work identifies catalytic and non-catalytic functions of Polκ in TLS and reveals important regulatory mechanisms underlying the unique domain architecture present at the C-terminal end of Y-family TLS polymerases. ### Competing Interest Statement The authors have declared no competing interest.