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，真核细胞利用各种称为检查点控制的监测机制。例如，在细胞复制过程中，细胞必须确保它们准确地复制了自己的DNA。为了应对精确复制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，激酶ATR在检测DNA复制问题并使细胞能够解决这些缺陷的机制中发挥着关键调节作用。该激酶的一个关键特征是，它在基因组扰动时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，一个被称为TRIP的结合伙伴帮助将ATR-ATIP复合体招募到单链DNA的RPA涂层区域，这是许多有害DNA损伤的特征。然而，ATR-ATrip复合体在与DNA上的RPA结合后仍然保持微弱的活性。在之后的某个时间点，ATR-ATrip与另一种名为TopBP1的蛋白质相互作用。这种结合导致ATR的激酶活性大量增加，并代表了触发检查点反应激活的许多步骤的顶峰。过去的研究表明，Rad9-Hus1-Rad1(9-1-1)复合体在调控TopBP1与ATR-Trip的相互作用中起着重要作用。然而，这一整体进程的许多方面仍然模糊不清。目前也不清楚ATR的激活是否仅通过这一途径在动物细胞中发生。我们最近观察到，Mre11-Rad50-Nbs1(MRN)复合体在激活ATR以响应异常DNA复制方面发挥了新的作用。MRN复合体最为人所知的是它在另一种检查点机制中的作用，即对双链DNA断裂(DSB)的反应。在即将到来的拨款期间，将进行各种研究，以阐明MRN与TopBP1和其他检查点控制蛋白合作促进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TopBP1在检查点响应过程中如何相互作用以及如何与复制叉子相互作用；以及(3)这些步骤如何最终导致ATR的激活。此外，还将在这些途径中寻找新的调节剂。这些研究将同时使用非洲爪哇卵提取液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。总体而言，这些研究将利用检查点信号的新视角，从而有望揭示保护基因组完整性的机制的原创性见解。这些信息对于理解细胞如何预防致癌突变和其他致病基因异常将是非常宝贵的。

 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.