Regulation of Telomere Maintenance in Fission Yeast

项目摘要/摘要 我们的实验室有兴趣了解真核细胞如何确保端粒的维持， 真核生物线性染色体的自然末端。进化保守的保护素和CST(CTc1/Cdc13- STN1-TEN1)复合体在端粒酶募集和端粒DNA保护中发挥重要作用 修复和检查点因素。端粒的稳定维持对于保持基因组的完整性和 防止可能导致肿瘤形成的不受欢迎的突变积累。端粒的调节 结构和端粒酶也影响衰老生物体的细胞增殖和组织维持。因此， 研究端粒和DNA损伤反应蛋白如何正确协作的基础机制研究 端粒维护应该提供必要的关键见解，以帮助设计更有效的治疗 针对肿瘤或其他与年龄相关的疾病的策略。我们建议的研究项目使用的是分裂酵母 裂殖酵母Pombe.裂解酵母端粒是人类端粒的良好模型，因为 参与端粒维持的蛋白质在分裂酵母和人类之间高度保守。 我们实验室和其他实验室的研究提供了详细的见解，说明了裂解酵母如何保护蛋白和Stn1-Ten1 确保裂解酵母中端粒的稳定维持。这些发现包括：(1)Tel1ATM/Rad3ATR_ Thr93上保护素亚单位Ccq1的依赖磷酸化通过以下途径促进端粒酶重新启动 促进Ccq1和端粒酶亚单位est1之间的相互作用，以及(2)另一个保护层的SUMO化 Lys242上的Tpz1ree亚基促进Stn1-Ten1在端粒上的募集并限制端粒的延伸。 在进化上保守的Tpz1残基也被发现能促进端粒酶。 激活和重新招募，进一步强调了裂解酵母调节端粒的保守性 和哺乳动物的庇护所。DNA聚合酶、端粒酶、保护素的时间结合模式分析 Stn1发现，庇护素亚基Rap1和Poz1以及Stn1-Ten1复合体促进及时解离 通过促进Pola的招募来完成端粒中的端粒酶的滞后链合成 端粒。对于目前的赠款申请，我们提议的实验将(1)识别和表征 允许CCQ1和POZ1促进Pola依赖的端粒保护的潜在调控机制(S) (目标1)，(2)确定新的Stn1-Ten1复合体的相互作用伙伴，并表征他们对 Stn1-Ten1复合体在端粒和非端粒位置的招募/保留(目标2)，以及(3)调查 Terra与Poly(A)+Terra表达调控对端粒Stn1-Ten1-Pola募集的影响 (目标3)。

Project Summary/Abstract Our laboratory is interested in understanding how eukaryotic cells ensure the maintenance of telomeres, the natural ends of linear eukaryotic chromosomes. Evolutionarily conserved shelterin and CST (CTC1/Cdc13- STN1-TEN1) complexes play essential roles in telomerase recruitment and protection of telomeres against DNA repair and checkpoint factors. Stable maintenance of telomeres is critical to preserve genomic integrity and prevent accumulation of undesired mutations that might lead to tumor formation. Regulation of telomere structures and telomerase also affect cell proliferation and tissue maintenance in aging organisms. Therefore, basic mechanistic studies investigating how telomere and DNA damage response proteins collaborate in proper telomere maintenance should provide critical insights necessary to help devise more effective treatment strategies against tumors or other age-related diseases. Our proposed research projects utilize fission yeast Schizosaccharomyces pombe. Fission yeast telomeres serve as a good model for human telomeres, since proteins involved in telomere maintenance are highly conserved between fission yeast and humans. Studies from our lab and others have provided detailed insights how fission yeast shelterin and Stn1-Ten1 ensure stable maintenance of telomeres in fission yeast. Those include findings that (1) Tel1ATM/Rad3ATR_ dependent phosphorylation of the shelterin subunit Ccq1 on Thr93 promotes telomerase recruitment by promoting interaction between Ccq1 and the telomerase subunit Est1, and (2) SUMOylation of another shelterin subunit Tpz1ree, on Lys242 facilitates Stn1-Ten1 recruitment to telomeres and limits telomere extension. Evolutionarily conserved "TEL patch" residues within Tpz1 have also been found to promote telomerase activation and recruitment, further highlighting the well conserved nature of telomere regulation by fission yeast and mammalian shelterin. Our analyses of temporal binding patterns for DNA polymerases, telomerase, shelterin and Stn1 found that shelterin subunits Rap1 and Poz1 and the Stn1-Ten1 complex promote timely dissociation of telomerase from telomeres by promoting recruitment of Pola to complete lagging strand synthesis at telomeres. For the current grant application, our proposed experiments will (1) identify and characterize underlying regulatory mechanism(s) that allow Ccq1 and Poz1 to promote Pola-dependent telomere protection (Aim 1), (2) identify new interaction partners of Stn1-Ten1 complex and characterize their contributions to recruitment/retention of Stn1-Ten1 complex at telomeres and non-telomeric sites (Aim 2), and (3) investigate how regulation of TERRA vs. poly(A)+ TERRA expression modulates Stn1-Ten1-Pola recruitment at telomeres (Aim 3).