PCNA is a master coordinator of many DNA-metabolic events. During DNA replication, the maturation of Okazaki fragments involves at least four DNA enzymes, all of which contain PCNA-interacting motifs. However, the temporal relationships and functional modulations between these PCNA-binding proteins are unclear. Here, we developed a strategy to purify endogenous PCNA-containing complexes from native chromatin, and characterized their structures using cryo-EM. Two structurally resolved classes (PCNA-FEN1 and PCNA-FEN1-RNaseH2 complexes) have captured a series of 3D snapshots for the primer-removal steps of Okazaki fragment maturation. These structures show that product release from FEN1 is a rate-liming step. Furthermore, both FEN1 and RNaseH2 undergo continuous conformational changes on PCNA that result in constant fluctuations in the bending angle of substrate DNA at the nick site, implying that these enzymes could regulate each other through conformational modulation of the bound DNA. The structures of the PCNA-FEN1-RNaseH2 complex confirm the toolbelt function of PCNA and suggests a potential unrecognized role of RNaseH2, as a dsDNA binding protein, in promoting the 5'-flap cleaving activity of FEN1.

中文翻译：

---

由 PCNA 结合的 FEN1 和 RNaseH2 介导的冈崎片段成熟的结构见解。


PCNA 是许多 DNA 代谢事件的主要协调者。在 DNA 复制过程中，冈崎片段的成熟涉及至少四种 DNA 酶，所有这些酶都包含 PCNA 相互作用基序。然而，这些 PCNA 结合蛋白之间的时间关系和功能调节尚不清楚。在这里，我们开发了一种从天然染色质中纯化含内源性 PCNA 的复合物的策略，并使用冷冻电镜表征了它们的结构。两个结构解析的类别 （PCNA-FEN1 和 PCNA-FEN1-RNaseH2 复合物） 捕获了一系列 3D 快照，用于冈崎片段成熟的引物去除步骤。这些结构表明，从 FEN1 释放产物是一个速率升限步骤。此外，FEN1 和 RNaseH2 在 PCNA 上都经历连续的构象变化，导致底物 DNA 在缺口位点的弯曲角度不断波动，这意味着这些酶可以通过结合 DNA 的构象调节来相互调节。PCNA-FEN1-RNaseH2 复合物的结构证实了 PCNA 的工具带功能，并表明 RNaseH2 作为 dsDNA 结合蛋白在促进 FEN1 的 5'-瓣裂解活性方面存在潜在的未识别作用。