Mechanisms of ATR Targeting and Regulation

描述（由申请人提供）：该提案的主要目标是描绘ATM和Rad 3相关蛋白质ATR的检查点信号功能所需的结构基序，并阐明ATR募集到DNA损伤位点的机制。ATR是磷酸肌醇3-激酶相关激酶基因超家族的成员，其被认为是哺乳动物DNA损伤反应的重要调节因子。ATR和相关蛋白ATM（共济失调-毛细血管扩张-突变）是大分子量蛋白激酶，其在信号级联的顶端起作用，所述信号级联调节细胞周期检查点激活、DNA修复和面对遗传损伤的转录反应。最近的进展已经导致ATR的检查点信号传导功能所需的细胞底物的鉴定，并指出ATR在S期期间发生的DNA损伤的信号传导中的关键作用。与ATR的基因组监视功能一致，最近的结果表明，ATR靶向DNA损伤位点和/或细胞暴露于DNA损伤剂或DNA复制抑制剂后停滞的复制叉。然而，在其保守的羧基末端催化结构域之外，ATR的DNA损伤信号传导功能所需的结构基序尚未被鉴定，ATR调节的潜在机制也未被阐明。在此背景下，我们提出：（1）描绘ATR的DNA复制检查点功能所需的功能基序;（2）映射和功能表征ATR中的核灶靶向结构域;（3）鉴定介导ATR靶向核灶的细胞蛋白。这些目标的实现将是理解ATR功能和调节机制的有力的第一步。我们希望从这些研究中获得的知识可以转化为对DNA损伤如何转化为细胞调节细胞以及最终如何在肿瘤发展过程中产生遗传不稳定性的更基本的理解。

DESCRIPTION (provided by applicant): The major goals of this proposal are to delineate the structural motifs that are required for the checkpoint signaling functions of the ATM and Rad3-related protein, ATR and to elucidate the mechanisms of ATR recruitment to sites of DNA damage. ATR is a member of the phosphoinositide 3-kinase-related kinase gene superfamily that has been implicated as an essential regulator of mammalian DNA damage responses. ATR and the related protein ATM (ataxia-telangiectasia-mutated) are large molecular mass protein kinases that function atop signaling cascades that regulate cell cycle checkpoint activation, DNA repair, and transcriptional responses in the face of genetic damage. Recent advances have led to the identification of cellular substrates that are required for the checkpoint signaling functions of ATR and have pointed toward a critical role for ATR in the signaling of DNA damage incurred during S phase. Consistent with genome surveillance functions of ATR, recent results have shown that ATR targets to sites of DNA damage and/or stalled replication forks following cellular exposure to DNA damaging agents or DNA replication inhibitors. However, outside of its conserved carboxyl-terminal catalytic domain, the structural motifs that are required for the DNA damage-signaling functions of ATR have not been identified, nor have the underlying mechanisms of ATR regulation been elucidated. Within this context, we propose to: (1) delineate functional motifs that are required for the DNA replication checkpoint functions of ATR; (2) map and functionally characterize a nuclear foci-targeting domain in ATR; and (3) identify the cellular protein(s) that mediate the targeting of ATR to nuclear foci. The accomplishment of these objectives will be a strong first step toward understanding the mechanisms of ATR function and regulation. We hope that knowledge gained from these studies can be translated into a more fundamental understanding of how DNA damage is converted into cell regulatory cells, and ultimately, how genetic instability arises during tumor development.