Graduate Program in Biochemistry, Molecular and Cell Biology, Weill Institute for Cell and Molecular Biology, Cornell University, 339 Weill Hall, Ithaca, New York 14853-7202, USA.
Mol Cell. 2010 Jul 30;39(2):300-6. doi: 10.1016/j.molcel.2010.06.019.
The DNA damage checkpoint kinase Mec1(ATR) is critical for maintaining the integrity of replication forks. Though it has been proposed to promote fork repair, the mechanisms by which Mec1 regulates DNA repair factors remain unclear. Here, we found that Mec1 mediates a key interaction between the fork protein Dpb11 and the DNA repair scaffolds Slx4-Rtt107 to regulate replication stress response. Dissection of the molecular basis of the interaction reveals that Slx4 and Rtt107 jointly bind Dpb11 and that Slx4 phosphorylation is required. Mutation of Mec1 phosphorylation sites in Slx4 disrupts its interaction with Dpb11 and compromises the cellular response to replisomes blocked by DNA alkylation. Multiple fork repair factors associate with Rtt107 or Slx4, supporting that Mec1-dependent assembly of the Rtt107-Slx4-Dpb11 complex functions to coordinate fork repair. Our results unveil how Mec1 regulates the Slx4 and Rtt107 scaffolds and establish a mechanistic link between DNA damage signaling and fork repair.
DNA 损伤检查点激酶 Mec1(ATR)对于维持复制叉的完整性至关重要。尽管它被提议促进叉修复，但 Mec1 调节 DNA 修复因子的机制仍不清楚。在这里，我们发现 Mec1 介导了叉蛋白 Dpb11 与 DNA 修复支架 Slx4-Rtt107 之间的关键相互作用，以调节复制应激反应。对相互作用的分子基础的剖析表明，Slx4 和 Rtt107 共同结合 Dpb11，并且 Slx4 的磷酸化是必需的。Mec1 中 Slx4 磷酸化位点的突变破坏了其与 Dpb11 的相互作用，并损害了细胞对 DNA 烷化剂阻断的复制体的反应。多个叉修复因子与 Rtt107 或 Slx4 相关联，支持 Mec1 依赖性 Rtt107-Slx4-Dpb11 复合物的组装功能，以协调叉修复。我们的研究结果揭示了 Mec1 如何调节 Slx4 和 Rtt107 支架，并在 DNA 损伤信号和叉修复之间建立了一种机制联系。
