[1] Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Radiation Oncology, Harvard Medical School, Boston, Massachusetts, USA.
Nat Struct Mol Biol. 2010 Mar;17(3):365-72. doi: 10.1038/nsmb.1769. Epub 2010 Feb 14.
Double-stranded DNA breaks (DSBs) induce a phosphorylation-mediated signaling cascade, but the role of phosphatases in this pathway remains unclear. Here we show that human protein phosphatase 4 (PP4) dephosphorylates replication protein A (RPA) subunit RPA2, regulating its role in the DSB response. PP4R2, a regulatory subunit of PP4, mediates the DNA damage-dependent association between RPA2 and the PP4C catalytic subunit. PP4 efficiently dephosphorylates phospho-RPA2 in vitro, and silencing PP4R2 in cells alters the kinetics and pattern of RPA2 phosphorylation. Depletion of PP4R2 impedes homologous recombination (HR) via inefficient loading of the essential HR factor RAD51, causing an extended G2-M checkpoint and hypersensitivity to DNA damage. Cells expressing phosphomimetic RPA2 mutants have a comparable phenotype, suggesting that PP4-mediated dephosphorylation of RPA2 is necessary for an efficient DNA-damage response. These observations provide new insight into the role and regulation of RPA phosphorylation in HR-mediated repair.
双链 DNA 断裂 (DSBs) 诱导磷酸化介导的信号级联反应，但磷酸酶在该途径中的作用尚不清楚。在这里，我们表明人类蛋白磷酸酶 4 (PP4) 去磷酸化复制蛋白 A (RPA) 亚基 RPA2，调节其在 DSB 反应中的作用。PP4R2 是 PP4 的调节亚基，介导 DSB 依赖性 RPA2 与 PP4C 催化亚基之间的关联。PP4 在体外有效去磷酸化磷酸化 RPA2，而细胞中 PP4R2 的沉默改变了 RPA2 磷酸化的动力学和模式。PP4R2 的耗竭通过必需的 HR 因子 RAD51 的加载效率降低来阻碍同源重组 (HR)，导致 G2-M 检查点延长和对 DNA 损伤的过度敏感。表达磷酸模拟 RPA2 突变体的细胞具有类似的表型，这表明 PP4 介导的 RPA2 去磷酸化对于有效的 DNA 损伤反应是必要的。这些观察结果为 RPA 磷酸化在 HR 介导的修复中的作用和调节提供了新的见解。
