Role of ATR in Cell Cycle Checkpoints

 描述（由申请人提供）：为了保持其基因组DNA的完整性，真核细胞利用称为检查点对照的各种监视机制。例如，在细胞复制过程中，细胞必须确保它们准确地复制了它们的DNA。为了科普精确复制DNA和纠正过程中出现的任何问题的挑战，细胞利用无数的调节蛋白。在脊椎动物中，ATR激酶在检测DNA复制问题并使细胞能够解决这些缺陷的机制中起着关键调节剂的作用。这种激酶的一个关键特征是它在基因组扰动后经历精确调节的激活。因此，这种激活涉及复杂的控制机制。例如，称为ATRIP的结合伴侣有助于将ATR-ATRIP复合物募集到单链DNA的RPA包被区域，这是许多有害DNA损伤的特征。然而，ATR-ATRIP复合物在与DNA上的RPA缔合后仍然保持弱活性。在之后的某个时刻，ATR-ATRIP与另一种称为TopBP 1的蛋白质相互作用。这种结合导致ATR的激酶活性大幅增加，并代表了触发检查点应答激活的许多步骤的高潮。过去的研究表明Rad 9-Hus 1-Rad 1（9-1-1）复合物在控制TopBP 1与ATR-ATRIP的相互作用中起作用。然而，这一总体进程的许多方面仍然模糊不清。目前还不清楚ATR的激活是否仅通过动物细胞中的这一途径发生。 我们最近观察到，Mre 11-Rad 50-Nbs 1（MRN）复合物在响应异常DNA复制的ATR激活中起着新的作用。MRN复合物最为人所知的是它在另一种类型的检查点机制中的作用，即对双链DNA断裂（DSB）的反应。在即将到来的资助期内，将开展各种研究，以阐明MRN与TopBP 1和其他检查点控制蛋白协同促进ATR激活的机制。将进行一系列系统的实验以探索：（1）MRN的核酸酶活性如何影响复制叉处的检查点诱导;（2）MRN和TopBP 1如何在检查点响应期间彼此相互作用并与复制叉相互作用;以及（3）这些步骤如何最终导致ATR的激活。此外，搜索将进行新的监管机构在这些途径。这些研究将使用非洲爪蟾卵提取物和人类组织培养细胞进行。这一战略将利用每个实验系统的互补优势。总的来说，这些研究将开拓检查点信号转导的新视角，从而有望揭示保护基因组完整性的机制的原始见解。这些信息对于理解细胞如何阻止促癌突变和其他诱发疾病的遗传异常非常宝贵。

 DESCRIPTION (provided by applicant): To preserve the integrity of their genomic DNA, eukaryotic cells utilize a variety of surveillance mechanisms known as checkpoint controls. For example, during cell duplication, cells must make sure that they have replicated their DNA accurately. To cope with the challenges of copying the DNA precisely and rectifying any problems that arise in the process, cells utilize a myriad of regulatory proteins. In vertebrates, the kinase ATR acts as a pivotal regulator in the mechanisms that detect problems with DNA replication and enable cells to address such defects. A key feature of this kinase is that it undergoes precisely regulated activation upon genomic perturbation. Accordingly, this activation involves elaborate control mechanisms. For example, a binding partner known as ATRIP helps to recruit the ATR-ATRIP complex to RPA-coated regions of single-stranded DNA, which are characteristic of numerous detrimental DNA lesions. However, the ATR- ATRIP complex still remains weakly active upon associating with RPA on the DNA. At some point thereafter, ATR-ATRIP interacts with another protein called TopBP1. This binding results in a massive increase in the kinase activity of ATR and represents the culmination of numerous steps that trigger activation of a checkpoint response. Past studies have indicated that the Rad9-Hus1-Rad1 (9-1-1) complex plays a role in controlling the interaction of TopBP1 with ATR-ATRIP. However, many aspects of this overall process have remained nebulous. It has also been unclear whether activation of ATR occurs solely through this route in animal cells. We have recently observed that the Mre11-Rad50-Nbs1 (MRN) complex plays a novel role in the activation of ATR in response to aberrant DNA replication. The MRN complex had been best known for its role in another type of checkpoint mechanism, namely, the response to double-stranded DNA breaks (DSBs). In the upcoming grant period, a variety of studies will be carried out to elucidate the mechanism by which MRN collaborates with TopBP1 and other checkpoint control proteins to promote the activation of ATR. A systematic series of experiments will be conducted to explore: (1) how the nuclease activity of MRN affects checkpoint induction at replication forks; (2) how MRN and TopBP1 interact with one another and with replication forks during the checkpoint response; and (3) how these steps ultimately lead to the activation of ATR. Moreover, searches will be undertaken for novel regulators in these pathways. These studies will be performed with both Xenopus egg extracts and human tissue culture cells. This strategy will capitalize upon the complementary advantages of each experimental system. Overall, these studies will exploit new perspectives on checkpoint signaling and hence promise to uncover original insights into the mechanisms that safegaurd genomic integrity. This information would be invaluable for understanding how cells forestall cancer-promoting mutations and other disease-inducing genetic abnormalities.