In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling
In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling
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DOI：
10.1074/jbc.m112.407825
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发表时间：
2012-10-19
影响因子：
4.8
通讯作者：
Sancar, Aziz
中科院分区：
文献类型：
--
作者：
Lindsey-Boltz, Laura A.;Reardon, Joyce T.;Sancar, Aziz
复制蛋白A（RPA）在DNA代谢中起重要作用，包括复制、检查点和修复。最近，我们描述了一个体外系统，其中ATR（共济失调毛细血管扩张症突变和Rad 3相关）对人Chk 1激酶的磷酸化依赖于与单链DNA结合的RPA。在这里，我们报告说，其他ATR目标，p53和Rad 17的磷酸化，具有相同的要求，RPA也在这个系统中磷酸化。在高p53或Rad 17浓度下，RPA磷酸化被抑制，并且在该系统中，具有拟磷酸化突变的RPA不能支持ATR激酶功能，而不可磷酸化的RPA突变体表现出完全活性。这些ATR底物的磷酸化取决于RPA对ATR和底物的募集至RPA-ssDNA复合物。最后，缺乏检查点功能的突变型RPA在核苷酸切除修复中表现出基本正常的活性，揭示了检查点和切除修复功能的RPA分离。
Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, wedescribed an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPAphosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.