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.

中文翻译：

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Exo1磷酸化抑制核酸外切酶活性，并防止rad53突变体中的叉子崩溃，而与14-3-3蛋白无关。

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。它的关键功能之一是防止Exo1依赖的前叉降解，并且Exo1被磷酸化以响应不同的遗传毒性剂。在存在复制压力的情况下，Exo1似乎受到几种翻译后修饰的调控，但尚不清楚检查点依赖性磷酸化对Exo1调控和叉子稳定性的具体贡献。我们在这里显示，Exo1磷酸化是响应DNA损伤或dNTP耗尽的Dun1独立和Rad53依赖，并且在两种情况下，Exo1在多个位点都被磷酸化。为了研究Exo1磷酸化与叉子稳定性之间的相关性，我们已经产生了磷酸化的模拟exo1等位基因，其挽救了rad53突变体中前叉塌陷的效率与exo1核酸酶死亡突变体或Exo1的缺失一样有效，理由是Rad53依赖的磷酸化是保持前叉稳定性的重要要求。我们还表明，这种挽救是独立于Bmh1-2的，至少在Exo1下调的情况下，14-3-3蛋白对于叉的稳定化是必不可少的。重要的是，我们的结果表明磷酸化特异性抑制5'至3'外切核酸酶的活性，这表明Exo1的这种活性而不是襟翼内切核酸酶是酶活性，是导致关卡突变体中停滞的复制叉崩溃的原因。认为Rad53依赖的磷酸化是维持叉子稳定性的市长要求。我们还表明，这种拯救是与Bmh1-2无关的，至少在Exo1下调时，14-14-3蛋白对于叉的稳定化是必不可少的。重要的是，我们的结果表明磷酸化特异性抑制5'至3'外切核酸酶的活性，这表明Exo1的这种活性而不是襟翼内切核酸酶是酶活性，是导致关卡突变体中停滞的复制叉崩溃的原因。认为Rad53依赖的磷酸化是维持叉子稳定性的市长要求。我们还表明，这种挽救是独立于Bmh1-2的，至少在Exo1下调的情况下，14-3-3蛋白对于叉的稳定化是必不可少的。重要的是，我们的结果表明磷酸化特异性抑制5'至3'外切核酸酶的活性，这表明Exo1的这种活性而不是襟翼内切核酸酶是酶活性，是导致关卡突变体中停滞的复制叉崩溃的原因。