Yao Guangyin, Wan Junhua, Liu Qizheng, Mu Chunhua, Wang Yue, Sang Jianli
Key Laboratory of Cell Proliferation and Regulation, College of Life Sciences, Beijing Normal University, Beijing, China.
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore
Biochem J. 2017 Mar 23;474(7):1293-1306. doi: 10.1042/BCJ20160889.
Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in cells. Phosphomimetic mutation of the three residues () impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the mutant also occurred in the mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in .
基因毒性应激会导致致病真菌中的DNA损伤、DNA复制停滞和丝状生长。DNA检查点激酶Rad53通过磷酸化执行应激反应的效应物进行关键调控。Rad53自身通过磷酸化被激活，通过去磷酸化被失活。先前的研究表明磷酸酶Pph3使Rad53去磷酸化。在此，我们使用质谱和诱变方法来鉴定酿酒酵母中Rad53上的Pph3去磷酸化位点。我们发现，在从甲磺酸甲酯（MMS）引起的DNA损伤恢复过程中，野生型细胞中被去磷酸化的丝氨酸残基351、461和477，在Pph3缺失的细胞中仍保持磷酸化状态。这三个残基的拟磷酸化突变（S351D、S461D和S477D）损害了Rad53的去磷酸化、细胞周期停滞的解除、两个Rad53效应物Dun1和Dbf4的去磷酸化，以及在从MMS诱导的DNA损伤恢复过程中从丝状生长到酵母生长的转变。在Pph3缺失突变体中观察到的表型在双特异性磷酸酶2（YVH1）缺失突变体中也出现。总之，我们的发现揭示了Pph3控制酿酒酵母中DNA损伤反应的分子机制。
