Regulation of Telomere Maintenance in Fission Yeast

裂殖酵母端粒维持的调控

• 批准号: 9269576
• 负责人: Toru Nakamura
• 金额: $ 36.19万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269576
• 关键词: Affect; Aging; Animal Model; Aplastic Anemia; Binding; Binding Sites; C-terminal; Cell Proliferation; Cells; Chromosomes; Collaborations; Complex; DNA; DNA Damage; DNA Repair; DNA damage checkpoint; DNA polymerase A; DNA-Directed DNA Polymerase; Data; Disease; Dissociation; Dyskeratosis Congenita; Ensure; Eukaryotic Cell; Failure; Fission Yeast; Functional disorder; Funding; Future; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Goals; Hamman-Rich syndrome; Health; Human; Inherited; Knowledge; Laboratories; Lead; Lysine; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Modeling; Mutation; Nucleoproteins; Organism; Pattern; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Translational Protein Processing; Prevention; Proteins; Recruitment Activity; Regulation; Research; Research Project Grants; Role; Serine; Single-Stranded DNA; Structure; Syndrome; Tail; Telomerase; Telomere Maintenance; Threonine; Time; Tissues; age related; base; chromosome fusion; ds-DNA; effective therapy; experimental study; genetic analysis; genome integrity; human disease; insight; interest; neoplastic cell; prevent; public health relevance; response; telomere; treatment strategy; tumor; tumor growth

 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.

 描述（由适用提供）：我们的实验室有兴趣了解真核细胞如何确保端粒的维持，即线性真核染色体的自然末端。进化构成庇护素和CST（CTC1/CDC13- STN1-TEN1）复合物在端粒募集和保护端粒中对DNA修复和检查点因子的保护起着重要作用。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突变的积累至关重要。端粒结构和端粒酶的调节还会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损伤反应蛋白如何在适当的端粒维持中进行协作的基础机械研究应提供必要的关键见解，以帮助制定针对肿瘤或其他年龄相关疾病的更有效的治疗策略。我们提出的研究项目使用裂变酵母菌酸果实pombe。裂变酵母端粒是人端粒的良好模型，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，裂变酵母菌细胞通过循环所有染色体而在严重的远程功能障碍中生存的能力为研究必需端粒维持因子的功能贡献提供了独特的机会，而不会受到细胞致死性的阻碍。在过去的资金期间，我们通过DNA损伤检查点激酶RAD3ATR和TEL1ATM确定了庇护素亚基CCQ1（THR93）的磷酸化是关键的翻译后修改，从而促进CCQ1与端粒化合物调节型监管型亚unitiit Est1 eSt1 tepymentase symentase ryments ryments rymitment syments playments py telements rymitment。 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 telomeres-dependent telomere elongation.我们对DNA聚合酶，端粒，庇护素和STN1的临时结合模式的详细分析还表明，RAP1，POZ1和STN1-TEN1通过及时促进POLα的延迟滞后链链的合成来促进端粒与端粒的及时分离。我们还通过质谱法成功鉴定了TPZ1TPP1和CCQ1上的许多磷酸化位点。在即将到来的资金期间，我们提出了实验，以定义TPZ1和CCQ1中其他磷酸化位点的功能意义（AIMS1-2），并研究Shelterin，STN1-TEN1和POLα如何协作以确保端粒维持（AIM3）。由于TPP1在滞后链合成和端粒合成和端粒招聘的磷酸化以及CST和庇护素依赖性的协调中也在哺乳动物细胞的端粒维持中起关键作用，因此成功完成所提出的实验的成功完成可能会对未来的哺乳动物端粒研究产生重大影响。