Zhu Songli, Fisher Laura A, Bessho Tadayoshi, Peng Aimin
Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, NE 68583, USA.
Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Nucleic Acids Res. 2017 Oct 13;45(18):10583-10594. doi: 10.1093/nar/gkx686.
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in mediating non-homologous end joining (NHEJ), a major repair pathway for DNA double-strand breaks (DSBs). The activation, function and dynamics of DNA-PKcs is regulated largely by its reversible phosphorylation at numerous residues, many of which are targeted by DNA-PKcs itself. Interestingly, these DNA-PKcs phosphorylation sites function in a distinct, and sometimes opposing manner, suggesting that they are differentially regulated via complex actions of both kinases and phosphatases. In this study we identified several phosphatase subunits as potential DSB-associated proteins. In particular, protein phosphatase 1 (PP1) is recruited to a DSB-mimicking substrate in Xenopus egg extracts and sites of laser microirradiation in human cells. Depletion of PP1 impairs NHEJ in both Xenopus egg extracts and human cells. PP1 binds multiple motifs of DNA-PKcs, regulates DNA-PKcs phosphorylation, and is required for DNA-PKcs activation after DNA damage. Interestingly, phosphatase 1 nuclear targeting subunit (PNUTS), an inhibitory regulator of PP1, is also recruited to DNA damage sites to promote NHEJ. PNUTS associates with the DNA-PK complex and is required for DNA-PKcs phosphorylation at Ser-2056 and Thr-2609. Thus, PNUTS and PP1 together fine-tune the dynamic phosphorylation of DNA-PKcs after DNA damage to mediate NHEJ.
DNA依赖性蛋白激酶催化亚基（DNA-PKcs）在介导非同源末端连接（NHEJ）中起关键作用，NHEJ是DNA双链断裂（DSB）的主要修复途径。DNA-PKcs的激活、功能和动力学在很大程度上受其众多残基的可逆磷酸化调节，其中许多残基是DNA-PKcs自身的作用靶点。有趣的是，这些DNA-PKcs磷酸化位点以独特的、有时甚至相反的方式发挥作用，这表明它们通过激酶和磷酸酶的复杂作用受到差异调节。在本研究中，我们鉴定了几个磷酸酶亚基作为潜在的与DSB相关的蛋白。特别是，蛋白磷酸酶1（PP1）在非洲爪蟾卵提取物中被募集到模拟DSB的底物上，在人类细胞中被募集到激光微照射位点。PP1的缺失会损害非洲爪蟾卵提取物和人类细胞中的NHEJ。PP1结合DNA-PKcs的多个基序，调节DNA-PKcs的磷酸化，并且是DNA损伤后DNA-PKcs激活所必需的。有趣的是，磷酸酶1核靶向亚基（PNUTS），一种PP1的抑制性调节因子，也被募集到DNA损伤位点以促进NHEJ。PNUTS与DNA-PK复合物结合，是DNA-PKcs在Ser-2056和Thr-2609处磷酸化所必需的。因此，PNUTS和PP1共同微调DNA损伤后DNA-PKcs的动态磷酸化以介导NHEJ。
