Abstract
Translesion DNA synthesis (TLS) is a cellular process that enables the bypass of DNA lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase κ (Polκ) has the distinctive 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 by Rev1 binding. On the one hand, Polκ is essential to replicate across a minor groove DNA lesion in a process that depends on PCNA ubiquitylation but is independent of Rev1. On the other hand, through its cooperative interaction with Rev1 and ubiquitylated PCNA, Polκ appears to stabilize 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's catalytic activity. Together, our work identifies catalytic and noncatalytic 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.