Meister Cindy, Wong Ronald P, Park Zhi-Hoon, Ulrich Helle D
Institute of Molecular Biology gGmbH, Ackermannweg 4, Mainz 55128, Germany.
Institute of Molecular Biology gGmbH, Ackermannweg 4, Mainz 55128, Germany.
DNA Repair (Amst). 2025 May;149:103842. doi: 10.1016/j.dnarep.2025.103842. Epub 2025 Apr 29.
Polyubiquitylation of the replication factor PCNA activates the replicative bypass of DNA lesions via an error-free pathway involving template switching. However, the mechanism by which the K63-linked polyubiquitin chains facilitate damage bypass is poorly understood. Intriguingly, stable fusions of linear ubiquitin oligomers to PCNA, designed as mimics of the native K63-linked chains, are not functional, while enzymatic modification of PCNA with linear chains supports template switching in budding yeast. To investigate the cause of this discrepancy, we have taken an alternative approach to identify the features of polyubiquitylated PCNA essential for activating damage bypass. We designed linear, non-cleavable ubiquitin constructs that can be recruited non-covalently to PCNA via a PIP motif. We found that these partially suppress the damage sensitivity and elevated spontaneous mutation rates of yeast strains defective in PCNA ubiquitylation. Genetic analysis confirms that this rescue is due to an activation of the template switching pathway. Surprisingly, even the recruitment of monoubiquitin units promotes activity in this setting. These observations suggest that the reversibility of ubiquitin's association with PCNA is more important than the actual linkage of the polyubiquitin chain. Thus, our study highlights the dynamic nature of ubiquitin signaling in the context of DNA damage bypass.
复制因子PCNA的多聚泛素化通过涉及模板转换的无差错途径激活DNA损伤的复制性绕过。然而，K63连接的多聚泛素链促进损伤绕过的机制尚不清楚。有趣的是，设计为天然K63连接链模拟物的线性泛素寡聚物与PCNA的稳定融合物没有功能，而用线性链对PCNA进行酶促修饰则支持芽殖酵母中的模板转换。为了研究这种差异的原因，我们采用了另一种方法来确定激活损伤绕过所必需的多聚泛素化PCNA的特征。我们设计了线性、不可切割的泛素构建体，其可以通过PIP基序非共价地募集到PCNA上。我们发现这些构建体部分抑制了PCNA泛素化缺陷的酵母菌株的损伤敏感性和自发突变率的升高。遗传分析证实这种拯救是由于模板转换途径的激活。令人惊讶的是，即使是单泛素单元的募集在这种情况下也能促进活性。这些观察结果表明，泛素与PCNA结合的可逆性比多聚泛素链的实际连接更为重要。因此，我们的研究突出了DNA损伤绕过背景下泛素信号传导的动态性质。
