Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA
Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA
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DOI：
10.1016/j.dnarep.2015.05.004
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发表时间：
2015-07-01
期刊：
影响因子：
3.8
通讯作者：
Lu, A. -Lien
中科院分区：
文献类型：
--
作者：
Hwang, Bor-Jang;Jin, Jin;Lu, A. -Lien
细胞周期检查点提供监视机制以激活DNA损伤反应，从而保持基因组完整性。异源三聚体Rad 9-Rad 1-Hus 1（9-1-1）钳是一种DNA损伤反应传感器，可以装载到DNA上。9-1-1通过与碱基切除修复（BER）中的几乎所有酶相互作用而参与BER。在这里，我们表明，个别9-1-1组件在BER由MYH DNA糖基化酶指导发挥不同的作用。Hus 1缺失突变体的分析表明，域间连接环（残基134-155）是MYH结合的关键决定因素。Hus 1的N-（残基1-146）和C-末端（残基147-280）的一半具有结构相似性，可以与MYH相互作用并刺激MYH。Husl（K136 A）突变体保留与MYH的物理相互作用，但不能刺激MYH糖基化酶活性。Hus 1的N-末端结构域，而不是C-末端的一半，也可以以中等亲和力结合DNA。在细菌中表达的完整Rad 9结合并微弱地刺激MYH。然而，Rad 9（1-266）（C-末端截短的Rad 9）可以刺激MYH活性并以高亲和力结合DNA，接近于异源三聚体9（1-266）-1-1复合物所显示的亲和力。相反，Rad 1在刺激MYH活性或与DNA结合方面的作用最小。最后，我们表明，优先招聘9（1-266）-1-1到5 '-凹陷的DNA底物是这种复合物的内在属性，是依赖于复合物的形成。总之，我们的研究结果提供了一个独特的贡献，由个别9-1-1亚基MYH-定向BER的基础上，在蛋白质-蛋白质相互作用和DNA结合事件的亚基不对称性的机械原理。(C)2015爱思唯尔B. V.保留所有权利。
Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Husl(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events. (C) 2015 Elsevier B.V. All rights reserved.