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Replication stress-induced Exo1 phosphorylation is mediated by Rad53/Pph3 and Exo1 nuclear localization is controlled by 14-3-3 proteins.
Cell Division ( IF 2.8 ) Pub Date : 2019-01-04 , DOI: 10.1186/s13008-018-0044-2
Nagaraja Chappidi 1 , Giuseppe De Gregorio 1 , Stefano Ferrari 1
Affiliation  

Background Mechanisms controlling DNA resection at sites of damage and affecting genome stability have been the subject of deep investigation, though their complexity is not yet fully understood. Specifically, the regulatory role of post-translational modifications in the localization, stability and function of DNA repair proteins is an important aspect of such complexity. Results Here, we took advantage of the superior resolution of phosphorylated proteins provided by Phos-Tag technology to study pathways controlling the reversible phosphorylation of yeast Exo1, an exonuclease involved in a number of DNA repair pathways. We report that Rad53, a checkpoint kinase downstream of Mec1, is responsible for Exo1 phosphorylation in response to DNA replication stress and we demonstrate a role for the type-2A protein phosphatase Pph3 in the dephosphorylation of both Rad53 and Exo1 during checkpoint recovery. Fluorescence microscopy studies showed that Rad53-dependent phosphorylation is not required for the recruitment or the release of Exo1 from the nucleus, whereas 14-3-3 proteins are necessary for Exo1 nuclear translocation. Conclusions By shedding light on the mechanism of Exo1 control, these data underscore the importance of post-translational modifications and protein interactions in the regulation of DNA end resection.

中文翻译:

复制应激诱导的 Exo1 磷酸化由 Rad53/Pph3 介导,而 Exo1 核定位由 14-3-3 蛋白控制。

背景 控制损伤部位 DNA 切除和影响基因组稳定性的机制一直是深入研究的主题,尽管它们的复杂性尚未完全了解。具体而言,翻译后修饰在 DNA 修复蛋白的定位、稳定性和功能中的调节作用是这种复杂性的一个重要方面。结果 在这里,我们利用 Phos-Tag 技术提供的磷酸化蛋白质的卓越分辨率来研究控制酵母 Exo1 可逆磷酸化的途径,酵母 Exo1 是一种参与许多 DNA 修复途径的核酸外切酶。我们报告 Rad53,Mec1 下游的检查点激酶,负责响应 DNA 复制应激的 Exo1 磷酸化,我们证明了 2A 型蛋白磷酸酶 Pph3 在检查点恢复期间在 Rad53 和 Exo1 的去磷酸化中的作用。荧光显微镜研究表明,从细胞核募集或释放 Exo1 不需要 Rad53 依赖性磷酸化,而 Exo1 核易位需要 14-3-3 蛋白。结论 通过阐明 Exo1 控制的机制,这些数据强调了翻译后修饰和蛋白质相互作用在 DNA 末端切除调控中的重要性。而 14-3-3 蛋白是 Exo1 核转位所必需的。结论 通过阐明 Exo1 控制的机制,这些数据强调了翻译后修饰和蛋白质相互作用在 DNA 末端切除调控中的重要性。而 14-3-3 蛋白是 Exo1 核转位所必需的。结论 通过阐明 Exo1 控制的机制,这些数据强调了翻译后修饰和蛋白质相互作用在 DNA 末端切除调控中的重要性。
更新日期:2020-04-22
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