Rev1–Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the cryogenic electron microscopic structure of the Saccharomyces cerevisiae Rev1–Polζ holocomplex in the act of DNA synthesis (3.53 Å). We discovered that a composite N-helix-BRCT module in Rev1 is the keystone of Rev1–Polζ cooperativity, interacting directly with the DNA template–primer and with the Rev3 catalytic subunit of Polζ. The module is positioned akin to the polymerase-associated domain in Y-family TLS polymerases and is set ideally to interact with PCNA. We delineate the full extent of interactions that the carboxy-terminal domain of Rev1 makes with Polζ and identify potential new druggable sites to suppress chemoresistance from first-line chemotherapeutics. Collectively, our results provide fundamental new insights into the mechanism of cooperativity between Rev1 and Polζ in TLS.

Rev1-Polz 依赖性 DNA 跨损伤合成 (TLS) 对于维持基因组完整性至关重要。为了阐明两种聚合酶在 TLS 中协作的机制，我们确定了DNA 合成过程中酿酒酵母Rev1-Polze 全复合物的低温电子显微镜结构 (3.53 Å)。我们发现 Rev1 中的复合 N 螺旋-BRCT 模块是 Rev1-Pol z 协同性的基石，直接与 DNA 模板引物以及 Pol z 的 Rev3 催化亚基相互作用。该模块的位置类似于 Y 家族 TLS 聚合酶中的聚合酶相关结构域，并且非常适合与 PCNA 相互作用。我们描绘了 Rev1 的羧基末端结构域与 Pol z 产生的相互作用的全部范围，并确定了潜在的新药物位点以抑制一线化疗药物的化疗耐药性。总的来说，我们的结果为 TLS 中 Rev1 和 Polz 之间的协作机制提供了基本的新见解。