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

描述（由申请人提供）：端粒结构的适当维持对于基因组的稳定遗传至关重要。各种检查点和DNA修复蛋白，包括进化上高度保守的检查点激酶Tel 1（ATM）和Rad 3（ATR），在端粒的稳定维持中发挥重要作用。然而，没有明确的机制作用，各种检查点和DNA修复蛋白在端粒的维护已经建立。我们研究的主要目标是了解检查点和DNA修复蛋白如何有助于端粒的维持。目前的建议将利用裂殖酵母粟酒裂殖酵母作为模型系统。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。端粒维持机制的失调已被发现是肿瘤发生中的关键事件，因此首先需要对各种蛋白质如何协作产生功能性端粒的机制见解来设计预防肿瘤发生的有效方法。为了稳定地维持，端粒必须履行两个主要功能。首先，端粒必须保护端粒DNA末端免于融合和降解。第二，端粒必须提供进入端粒酶的途径，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允许端粒酶募集的更易接近状态的动态切换。我们假设，检查点和DNA修复蛋白被招募到端粒在细胞周期调节的方式，引发端粒结构和端粒蛋白组成的适当变化。该模型将在目标1中直接测试。此外，新开发的裂殖酵母报告菌株将被用来寻找目标2中Rad 3和Tel 1激酶的潜在端粒靶点。我们对MRN（Mre 11-Rad 50-Nbs 1）复合物突变体的初步研究表明，与将Tel 1募集到其他DNA断裂不同，将Tel 1募集到端粒不需要Tel 1-MRN相互作用。因此，将在目标3中尝试理解Tel 1向端粒的MRN独立募集的机制基础。

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.