Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
Institute of Molecular Biology gGmbH (IMB), Ackermannweg 4, D-55128 Mainz, Germany.
Nucleic Acids Res. 2020 Apr 6;48(6):3042-3052. doi: 10.1093/nar/gkaa053.
Ubiquitylation of the eukaryotic sliding clamp, PCNA, activates a pathway of DNA damage bypass that facilitates the replication of damaged DNA. In its monoubiquitylated form, PCNA recruits a set of damage-tolerant DNA polymerases for translesion synthesis. Alternatively, modification by K63-linked polyubiquitylation triggers a recombinogenic process involving template switching. Despite the identification of proteins interacting preferentially with polyubiquitylated PCNA, the molecular function of the chain and the relevance of its K63-linkage are poorly understood. Using genetically engineered mimics of polyubiquitylated PCNA, we have now examined the properties of the ubiquitin chain required for damage bypass in budding yeast. By varying key parameters such as the geometry of the junction, cleavability and capacity for branching, we demonstrate that either the structure of the ubiquitin-ubiquitin junction or its dynamic assembly or disassembly at the site of action exert a critical impact on damage bypass, even though known effectors of polyubiquitylated PCNA are not strictly linkage-selective. Moreover, we found that a single K63-junction supports substantial template switching activity, irrespective of its attachment site on PCNA. Our findings provide insight into the interrelationship between the two branches of damage bypass and suggest the existence of a yet unidentified, highly linkage-selective receptor of polyubiquitylated PCNA.
泛素化的真核滑动夹 PCNA 激活了一条 DNA 损伤旁路途径，促进了受损 DNA 的复制。在其单泛素化形式中，PCNA 招募了一组耐受损伤的 DNA 聚合酶进行跨损伤合成。或者，通过 K63 连接的多泛素化修饰触发涉及模板切换的重组过程。尽管已经鉴定出与多泛素化 PCNA 优先相互作用的蛋白质，但链的分子功能及其 K63 连接的相关性仍知之甚少。使用遗传工程模拟的多泛素化 PCNA，我们现在已经研究了在芽殖酵母中进行损伤旁路所必需的泛素链的性质。通过改变关键参数，如连接点的几何形状、可切割性和分支能力，我们证明，无论是泛素-泛素连接点的结构，还是其在作用部位的动态组装或拆卸，都会对损伤旁路产生关键影响，尽管已知的多泛素化 PCNA 效应物不是严格的连接选择性。此外，我们发现单个 K63 连接点支持大量的模板切换活性，而与其在 PCNA 上的附着位点无关。我们的研究结果深入了解了损伤旁路的两个分支之间的相互关系，并暗示存在一个尚未识别的、高度连接选择性的多泛素化 PCNA 受体。
