Roles of Checkpoint and DNA Repair Proteins in Fission Yeast Telomere Maintenance

描述（申请人提供）：端粒结构的适当维持对基因组的稳定遗传至关重要。多种检查点和DNA修复蛋白，包括进化上高度保守的检查点激酶Tel1 （ATM）和Rad3 (ATR)，在端粒的稳定维持中发挥重要作用。然而，各种检查点蛋白和DNA修复蛋白在端粒维持中的机制作用尚未明确。我们研究的主要目标是了解检查点和DNA修复蛋白如何促进端粒维护。目前的建议将利用分裂酵母裂糖酵母作为模型系统。裂变酵母和人类之间高度保守的DNA损伤反应和端粒维持机制应该有助于推断我们的发现，以建立可测试的人类细胞模型。端粒维持机制的解除管制已被发现是肿瘤发生的关键事件，因此，首先需要了解各种蛋白质如何协同产生功能性端粒的机制，以设计有效的预防肿瘤发生的方法。为了稳定地维持，端粒必须完成两个主要功能。首先，端粒必须保护端粒DNA末端免受融合和降解。其次，端粒必须提供端粒酶，以防止DNA复制后端粒DNA的丢失。因此，端粒必须经历从高度保护状态到更容易接近的状态的动态转换，从而允许端粒酶的招募。我们假设检查点蛋白和DNA修复蛋白以细胞周期调节的方式被募集到端粒，从而触发端粒结构和端粒蛋白组成的适当变化。该模型将在Aim 1中直接进行测试。此外，新开发的裂变酵母报告菌株将用于寻找Aim 2中Rad3和Tel1激酶的潜在端粒靶点。我们对MRN （Mre11-Rad50-Nbs1）复合物突变体的初步研究表明，与向其他DNA断裂募集Tel1不同，将Tel1募集到端粒不需要Tel1-MRN相互作用。因此，Aim 3将尝试了解不依赖mrn的Tel1向端粒募集的机制基础。

DESCRIPTION (provided by applicant): Proper maintenance of telomere structure is crucial for stable inheritance of the genome. Various checkpoint and DNA repair proteins, including evolutionarily highly conserved checkpoint kinases Tel1 (ATM) and Rad3 (ATR), play important roles in stable maintenance of telomeres. However, no clear mechanistic roles for various checkpoint and DNA repair proteins in telomere maintenance have been established. Major goal of our research is to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. The current proposal will utilize fission yeast Schizosaccharomyces pombe as a model system. Highly conserved DNA damage responses and telomere maintenance mechanisms between fission yeast and humans should be helpful in extrapolating our findings to build testable models for human cells. Deregulation of telomere maintenance mechanisms has been found to be a key event in tumorigenesis, thus mechanistic insights on how various proteins collaborate to generate functional telomeres are first needed to devise effective methods for preventing tumorigenesis. In order to be stably maintained, telomeres must fulfill two major functions. First, telomeres must protect telomeric DNA ends from fusions and degradation. Second, telomeres must provide access to telomerase to prevent loss of telomeric DNA after DNA replication. Thus, telomeres must undergo dynamic switches from the highly protected state to the more accessible state that allows recruitment of telomerase. We hypothesize that checkpoint and DNA repair proteins are recruited to telomeres in a cell cycle-regulated manner to trigger appropriate changes in telomere structure and telomere protein composition. This model will be directly tested in Aim 1. Additionally, newly developed fission yeast reporter strains will be utilized to search for potential telomere targets of Rad3 and Tel1 kinases in Aim 2. Our preliminary studies with MRN (Mre11-Rad50-Nbs1) complex mutants have suggested that recruitment of Tel1 to telomeres does not require Tel1-MRN interaction unlike recruitment of Tel1 to other DNA breaks. Therefore, attempts will be made in Aim 3 to understand the mechanistic basis for the MRN-independent recruitment of Tel1 to telomeres.