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.

跨损伤 DNA 合成 (TLS) 是一种细胞过程，能够绕过 DNA 复制过程中遇到的 DNA 损伤，并且正在成为化疗的主要目标。在脊椎动物 DNA 聚合酶中，聚合酶 κ (Polκ) 具有在体外绕过小沟 DNA 加合物的独特能力。然而，细胞克服主沟 DNA 加合物也需要 Polκ，但这一要求的基础尚不清楚。在这里，我们将人类细胞中的 CRISPR 碱基编辑器筛选技术与爪蟾卵提取物中确定的 DNA 损伤的 TLS 分析相结合，以揭示 Polκ 在损伤旁路过程中的功能和调节。引人注目的是，我们发现 Polκ 在 TLS 期间有两个主要功能，这些功能受到 Rev1 结合的差异调节。一方面，Polκ 对于跨越小沟 DNA 损伤进行复制至关重要，该过程依赖于 PCNA 泛素化，但独立于 Rev1。另一方面，通过与 Rev1 和泛素化 PCNA 的协同相互作用，Polκ 似乎可以稳定 DNA 上的 Rev1-Pol z 延伸复合物，从而允许延伸穿过主沟 DNA 损伤和无碱基位点，这一过程独立于 Polκ 的催化活性。我们的工作共同确定了 Polκ 在 TLS 中的催化和非催化功能，并揭示了 Y 家族 TLS 聚合酶 C 末端独特结构域结构背后的重要调控机制。