Bonilla Carla Yaneth, Melo Justine Amy, Toczyski David Paul
Department of Biochemistry and Biophysics, Cancer Research Institute, University of California, San Francisco, 2340 Sutter Street, San Francisco, CA 94115, USA.
Mol Cell. 2008 May 9;30(3):267-76. doi: 10.1016/j.molcel.2008.03.023.
Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether colocalization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially colocalized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation, and metaphase delay, demonstrating that colocalization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to establish that CDK functions in the checkpoint pathway downstream of damage processing and checkpoint protein recruitment. This CDK dependence is likely, at least in part, through Rad9, since mutation of CDK consensus sites compromised its checkpoint function.
先前对酿酒酵母DNA损伤检查点的研究表明，有两个复合物可独立感知DNA损伤：激酶Mec1-Ddc2和类PCNA的9-1-1复合物。为了测试这些组分的共定位是否足以激活检查点，我们将这些检查点蛋白与LacI阻遏物融合，并通过在含有Lac操纵子阵列的细胞中表达这些融合蛋白来人为地使其共定位。我们观察到Rad53和Rad9磷酸化、Sml1降解以及中期延迟，这表明在没有DNA损伤的情况下，这些传感器的共定位足以激活检查点。我们的拴系系统使我们能够确定CDK在损伤处理和检查点蛋白募集的下游检查点途径中发挥作用。这种对CDK的依赖性可能至少部分是通过Rad9实现的，因为CDK共有位点的突变损害了其检查点功能。
