Two checkpoint complexes are independently recruited to sites of DNA damage in vivo.
Two checkpoint complexes are independently recruited to sites of DNA damage in vivo.
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DOI：
10.1101/gad.903501
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发表时间：
2001-11
影响因子：
10.5
通讯作者：
Justine A Melo;J. Cohen;D. Toczyski
中科院分区：
文献类型：
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作者：
Justine A Melo;J. Cohen;D. Toczyski
Ddc 1/Rad 17/Mec 3复合物和Rad 24分别是与复制因子PCNA和RF-C具有有限同源性的DNA损伤检查点组分，表明这些因子通过直接“感知”DNA损伤来促进检查点激活。然而，Mec 1激酶磷酸化检查点蛋白Ddc 2，以响应在所有其他已知的检查点蛋白的情况下的损伤，这表明Mec 1和/或Ddc 2可能作为DNA损伤的初始传感器。在本文中，我们表明，Ddc 1或Ddc 2融合到绿色荧光蛋白定位到一个单一的亚核焦点以下的内切核酸酶断裂。其他形式的损伤导致更多数量的Ddc 1-GFP或Ddc 2-GFP灶，与产生的损伤位点的数量相关，表明Ddc 1和Ddc 2都被募集到DNA损伤位点。有趣的是，Ddc 2定位在mec 1细胞中被严重废除，但不需要其他已知的检查点基因，而Ddc 1定位需要Rad 17，Mec 3和Rad 24，但不需要Mec 1。因此，Ddc 1和Ddc 2通过独立的机制识别DNA损伤。这些数据支持一种模型，其中在DNA损伤位点组装多个检查点复合物刺激检查点激活。此外，我们发现，虽然Ddc 1仍然强烈本地化检查点适应后，许多细胞核只包含暗淡的Ddc 2-GFP焦点，这表明Ddc 2的本地化可能会下调细胞分裂的恢复过程中。最后，定位于损伤位点的检查点蛋白的可视化作为分析活细胞中DNA损伤的有用工具。
The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.