Analysis of a DNA Damage-Inducible Checkpoint Complex

描述（由申请人提供）：我们基因组的完整性受到复制过程中产生的错误、代谢产生的活性氧、自发脱嘌呤以及损伤DNA的内源性和外源性因子的持续攻击。复制应激和DNA损伤激活检查点信号通路，通过协调DNA修复、基因转录和细胞周期阻滞来帮助维持基因组稳定性。关键检查点信号传导途径是Atr-Chkl信号传导途径，其被复制应激和其他类型的DNA损伤激活，并且在维持基因组稳定性中起重要作用。Atr-Chkl信号传导途径中的关键组分是Rad 9、Husl和Radl，其形成Rad 9- Husl-Radl（911）复合物，并且是最佳Chkl活化以及对DNA损伤的其他细胞应答所需的。911复合物形成了一个类似PCNA的夹子，在损伤部位的DNA周围加载。尽管钳装载在DNA周围并且是最佳Chkl活化所需的，但钳在Chkl活化和协调其他细胞事件中的实际作用仍然未知。在本提案的目标1中，我们已经鉴定了一种新的Rad 9相互作用蛋白，其结合Rad 9的C末端，这是Chkl活化所需的区域，并且我们建议检查这种相互作用蛋白对Rad 9功能和Chkl活化的作用。在目标2中，我们描述了Rad 9 B，一个新的Rad 9 paramount，并证明Rad 9，Rad 9 B，Husl，HuslB（一个Husl paramount），和Radl combinatorially组装成不同的夹具。然后，我们建议检查的作用，每个亚基在细胞反应的一个面板的遗传毒性剂，以确定每个911亚基是否有独特的和/或重叠的作用。在目的3中，我们表明，911复合物与translesion DNA聚合酶相互作用，我们提出的实验，以检查911亚基在translesion聚合酶功能的作用。总的来说，这些研究将为911复合物如何指导调节检查点激活，细胞存活和基因组稳定性的下游事件提供新的见解。

DESCRIPTION (provided by applicant): The integrity of our genomes is under continual assault from errors generated during replication, metabolically produced reactive oxygen species, spontaneous depurination, and endogenous and exogenous agents that damage DNA. Replication stress and DNA damage activate checkpoint signaling pathways that help maintain gemonic stability by orchestrating DNA repair, gene transcription, and cell cycle arrest. A critical check point signaling pathway is the Atr-Chkl signaling pathway, which is activated by replication stress and other types of DNA damage and plays an important role in maintaining genomic stability. Key partcipants in the Atr-Chkl signaling pathway are Rad9, Husl, and Radl, which form a Rad9- Husl-Radl (911) complex, and are required for optimal Chkl activation as well as other cellular responses to DNA damage. The 911 complex forms a PCNA-like clamp that is loaded around DNA at sites of damage. Although the clamp is loaded around DNA and is required for optimal Chkl activation, the clamp's actual role in Chkl activation and orchestrating other cellular events remains unknown. In Aim 1 of this proposal, we have identified a novel Rad9-interacting protein that binds the C terminus of Rad9, a region required for Chkl activation, and we propose to examine the role of this interacting protein on Rad9 function and Chkl activation. In Aim 2, we describe Rad9B, a new Rad9 paralog, and demonstrate that Rad9, Rad9B, Husl, HuslB (a Husl paralog), and Radl combinatorially assemble into different clamps. We then propose to examine the roles of each subunit in cellular responses to a panel of genotoxic agents to identify whether each 911 subunit has unique and/or overlapping roles. In Aim 3, we show that the 911 complex interacts with a translesion DNA polymerase and we propose experiments to examine the role of 911 subunits in translesion polymerase function. Collectively, these studies will provide new insights into how the 911 complexes direct downstream events that regulate checkpoint activation, cell survival, and genomic stability.