El-Shemerly Mahmoud, Hess Daniel, Pyakurel Aswin K, Moselhy Said, Ferrari Stefano
Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Nucleic Acids Res. 2008 Feb;36(2):511-9. doi: 10.1093/nar/gkm1052. Epub 2007 Nov 29.
Nucleases play important roles in DNA synthesis, recombination and repair. We have previously shown that human exonuclease 1 (hEXO1) is phosphorylated in response to agents stalling DNA replication and that hEXO1 consequently undergoes ubiquitination and degradation in a proteasome-dependent manner. In the present study, we have addressed the identity of the pathway transducing stalled-replication signals to hEXO1. Using chemical inhibitors, RNA interference, ATM- and ATR-deficient cell lines we have concluded that hEXO1 phosphorylation is ATR-dependent. By means of mass spectrometry, we have identified the sites of phosphorylation in hEXO1 in undamaged cells and in cells treated with hydroxyurea (HU). hEXO1 is phosphorylated at nine basal sites and three additional sites are induced by HU treatment. Analysis of single- and multiple-point mutants revealed that mutation to Ala of the three HU-induced sites of phosphorylation partially rescued HU-dependent degradation of hEXO1 and additionally stabilized the protein in non-treated cells. We have raised an antibody to pS(714), an HU-induced site of the S/T-Q type, and we provide evidence that S(714) is phosphorylated upon HU but not IR treatment. The antibody may be a useful tool to monitor signal transduction events triggered by stalled DNA replication.
核酸酶在DNA合成、重组和修复过程中发挥着重要作用。我们之前已经表明，人类核酸外切酶1（hEXO1）会响应使DNA复制停滞的因素而发生磷酸化，并且hEXO1随后会以蛋白酶体依赖的方式经历泛素化和降解。在本研究中，我们探讨了将复制停滞信号传导至hEXO1的信号通路的身份。使用化学抑制剂、RNA干扰、ATM和ATR缺陷细胞系，我们得出结论，hEXO1磷酸化是依赖ATR的。通过质谱分析，我们确定了未受损细胞和用羟基脲（HU）处理的细胞中hEXO1的磷酸化位点。hEXO1在九个基础位点发生磷酸化，另外三个位点是由HU处理诱导产生的。对单点和多点突变体的分析表明，将三个HU诱导的磷酸化位点突变为丙氨酸可部分挽救hEXO1依赖HU的降解，并额外稳定未处理细胞中的蛋白质。我们制备了针对pS(714)（一种HU诱导的S/T-Q型位点）的抗体，并且我们提供证据表明S(714)在HU处理后而非IR处理后发生磷酸化。该抗体可能是监测由停滞的DNA复制触发的信号转导事件的有用工具。
