Molecular Mechanism on the Regulation of Telomere Structure by Checkpoint Factors

检查点因子调控端粒结构的分子机制

• 批准号: 16570007
• 负责人: MATSUURA Akira
• 金额: $ 2.37万
• 依托单位: National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16570007/
• 关键词: telomere; checkpoint; ATM-related proteins; cell cycle; telomerase; DNA double-strand repair; クロマチン免疫沈降法; DNA末端修復; MRX複合体; クロマチン免疫沈隆法; telomere; checkpoint; ATM-related proteins; cell cycle; telomerase; DNA double-strand repair; クロマチン免疫沈降法; DNA末端修復; MRX複合体; クロマチン免疫沈隆法

The telomere is an essential nucleoprotein structure that protects the ends of eukaryotic linear chromosomes. The telomere DNA consists of a unique repeat that is synthesized by telomerase, which is a specialized reverse transcriptase. Due to problems with end-replication, cells that lack telomerase activity are unable to maintain the original telomere length after replication. Therefore, successive rounds of replication in telomerase-deficient cells generate unstable telomere-less chromosomes, which subsequently cause impairment of cell proliferation.The maintenance of telomeric DNA requires additional factors, at least in yeasts, including a wide range of proteins that are involved in DNA metabolism. Using the chromain immunoprecipitation assay, we showed that Tel1p and Mec1p, PIKK family proteins in Saccharomyces cerevisiae, were recruited to the telomeres at specific times in the cell cycle, in a mutually exclusive manner. Mec1p interacted with the telomeres during late S phase, and this interaction required its own kinase activity, which peaked during the S phase. Moreover, we showed that the MRX (Mre11-Rad50-Xrs2) complex, which functions in DNA double-strand break repair, was recruited to the telomeres in the late S phase, and that its loading was related to the extension of single-stranded telomere tails during this period. MRX was required for the late S phase-specific recruitment of Mec1 (ATR) to the telomeres. Mec1, in turn, contributed to the assembly of the telomerase regulators Cdc13 and Est1 at the telomere ends. Based on these results, we proposed a model for the hierarchical assembly of telomere replication proteins in the late S phase, which involves triggering by the loading of MRX onto the chromosome termini. The recruitment of DNA repair-related proteins to the telomeres at particular times in the cell cycle suggests that the normal terminus of a chromosome is recognized as a DNA double-strand break once per cell cycle.

端粒是一种必不可少的核蛋白结构，可保护真核线性染色体的末端。端粒DNA由一个独特的重复组成，该重复由端粒酶合成，端粒酶是一种专门的逆转录酶。由于最终复制问题，缺乏端粒酶活性的细胞无法在复制后保持原始的端粒长度。因此，端粒酶缺陷细胞的连续复制回合会产生不稳定的无端粒染色体，随后会导致细胞增殖受损。端粒DNA的维持需要其他因素，至少在酵母中，包括涉及DNA代谢的广泛蛋白质。使用Chromain免疫沉淀测定法，我们表明酿酒酵母中的Tel1p和MeC1p，Pikk家族蛋白在细胞周期的特定时间招募到端粒，以相互的独特方式招募到端粒。 MEC1P在S阶段后期与端粒相互作用，这种相互作用需要其自身的激酶活性，在S期达到峰值。此外，我们证明了在DNA双链破裂修复中起作用的MRX（MRE11-RAD50-XRS2）复合物在S阶段后期招募到端粒，并且其负载与此期间的单链端粒尾部的扩展有关。 M MRX是对端粒的MEC1（ATR）招募的晚期特异性募集。反过来，MEC1促成了端粒末端的端粒酶调节剂CDC13和EST1的组装。基于这些结果，我们提出了一个在S阶段晚期端粒复制蛋白层次组件的模型，该模型涉及通过将MRX载入到染色体末端的触发。在细胞周期的特定时间，将DNA修复相关蛋白募集到端粒中表明，染色体的正常末端被认为是每个细胞周期一次DNA双链断裂。