Regulation of Telomere Maintenance in Fission Yeast

 描述（由申请人提供）：我们的实验室有兴趣了解真核细胞如何确保端粒（线性真核染色体的天然末端）的维持。进化上保守的shelterin和CST（CTC 1/Cdc 13-STN 1-TEN 1）复合物在端粒酶募集和保护端粒免受DNA修复和检查点因子的影响中起重要作用。端粒的稳定维持对于保持基因组完整性和防止可能导致肿瘤形成的不期望的突变的积累至关重要。端粒结构和端粒酶的调节也影响衰老生物体中的细胞增殖和组织维持。因此，研究端粒和DNA损伤反应蛋白如何在适当的端粒维持中合作的基本机制研究应该提供必要的关键见解，以帮助设计更有效的治疗策略来对抗肿瘤或其他年龄相关疾病。我们建议的研究项目利用裂殖酵母粟酒裂殖酵母。裂变酵母端粒作为人类端粒的一个很好的模型，因为参与端粒维持的蛋白质在裂变酵母和人类之间高度保守。此外，分裂酵母细胞通过使其所有染色体环化而存活于严重端粒功能障碍的能力提供了独特的机会来研究必需的端粒维持因子的功能贡献，而不受细胞致死性的阻碍。 在过去的资助期间，我们确定了由DNA损伤检查点激酶Rad 3ATR和Tel 1ATM在Thr 93（Thr 93）处磷酸化的shelterin亚基Ccq 1作为关键的翻译后修饰，其促进Ccq 1和端粒酶调节亚基Est 1之间的相互作用以允许端粒酶募集。此外，我们证明了裂殖酵母shelterin和Stn 1-Ten 1复合物相互作用，并且shelterin亚基Tpz 1 TPP 1在赖氨酸-242（Lys 242）处的SUMO化促进shelterin-Stn 1-Ten 1相互作用，以允许Stn 1-Ten 1在端粒处有效积累并限制端粒依赖性端粒延长。我们对DNA聚合酶、端粒酶、shelterin和Stn 1的时间结合模式的详细分析也表明，Rap 1、Poz 1和Stn 1-Ten 1通过促进Polα的及时募集以完成端粒处的滞后链合成来促进端粒酶从端粒的及时解离。我们还通过质谱法成功地鉴定了Tpz 1 TPP 1和Ccq 1上的许多磷酸化位点。在即将到来的资助期内，我们提出了实验来确定Tpz 1和Ccq 1中额外磷酸化位点的功能意义（Aims 1 -2），并研究shelterin，Stn 1-Ten 1和Polα如何合作以确保端粒的维持（Aim 3）。由于TPP 1也是高度磷酸化的，并且滞后链合成和端粒酶募集的CST-和shelterin-依赖性协调在哺乳动物细胞中的端粒维持中也起关键作用，因此所提出的实验的成功完成将可能对未来的哺乳动物端粒研究产生重大影响。

 DESCRIPTION (provided by applicant): 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. In addition, the ability of fission yeast cells to survive severe telomer dysfunction by circularizing all their chromosomes provides unique opportunities to study functional contributions of essential telomere maintenance factors, without being hindered by cell lethality. During the past funding period, we identified phosphorylation of the shelterin subunit Ccq1 at Threonine-93 (Thr93) by DNA damage checkpoint kinases Rad3ATR and Tel1ATM as the critical post-translational modification that promotes interaction between Ccq1 and the telomerase regulatory subunit Est1 to allow telomerase recruitment. Furthermore, we demonstrated that fission yeast shelterin and Stn1-Ten1 complexes interact, and that SUMOylation of the shelterin subunit Tpz1TPP1 at Lysine-242 (Lys242) facilitates shelterin-Stn1-Ten1 interaction to allow efficient accumulation of Stn1-Ten1 at telomeres and to limit telomerase-dependent telomere elongation. Our detailed analyses of temporal binding patterns for DNA polymerases, telomerase, shelterin and Stn1 also demonstrated that Rap1, Poz1 and Stn1-Ten1 promote timely dissociation of telomerase from telomeres by promoting timely recruitment of Polα to complete lagging strand synthesis at telomeres. We have also successfully identified numerous phosphorylation sites on Tpz1TPP1 and Ccq1 by mass spectrometry. For the upcoming funding period, we propose experiments to define the functional significance of additional phosphorylation sites in Tpz1 and Ccq1 (Aims1-2) and investigate how shelterin, Stn1-Ten1 and Polα collaborate to ensure telomere maintenance (Aim3). Since TPP1 is also highly phosphorylated and CST- and shelterin- dependent coordination of lagging strand synthesis and telomerase recruitment also play critical roles in telomere maintenance in mammalian cells, successful completion of the proposed experiments will likely have major impacts on future mammalian telomere studies.