Instituto de Biología Funcional y Genómica (CSIC/USAL), Campus Miguel de Unamuno, Salamanca 37007, Spain.
Departamento de Bioquímica y Biología Molecular, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS) - Instituto de Investigación Sanitaria (IDIS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Nucleic Acids Res. 2020 Apr 6;48(6):3053-3070. doi: 10.1093/nar/gkaa054.
The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1-2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.
S 期检查点对于在威胁 DNA 复制的条件下维持基因组稳定性至关重要。其关键功能之一是防止 Exo1 依赖性叉降解，并且 Exo1 在响应不同的遗传毒性剂时被磷酸化。在复制应激存在的情况下，Exo1 似乎受到几种翻译后修饰的调节，但检查点依赖性磷酸化对 Exo1 控制和叉稳定性的具体贡献尚不清楚。我们在这里表明，Exo1 的磷酸化在存在复制压力的情况下是 Dun1 非依赖性和 Rad53 依赖性的，但在这两种情况下，Exo1 在多个位点被类似地磷酸化。为了研究 Exo1 磷酸化与叉稳定性之间的相关性，我们生成了磷酸模拟的 exo1 等位基因，这些等位基因在 rad53 突变体中有效地挽救了叉崩溃，就像 exo1 核酸酶失活突变体或缺乏 Exo1 一样，这表明 Rad53 依赖性磷酸化是维持叉稳定性的主要要求。我们还表明，这种挽救与 Bmh1-2 无关，这表明 14-3-3 蛋白对于叉稳定是可有可无的，至少在 Exo1 下调时是这样。重要的是，我们的结果表明磷酸化特异性抑制 5'到 3'外切核酸酶活性，表明 Exo1 的这种活性而不是 flap-内切核酸酶，是导致检查点突变体中停滞复制叉崩溃的酶促活性。
