Role of human CST in preventing telomere loss

描述（由申请人提供）：端粒是染色体的物理末端，通过防止染色体受损来保护基因组稳定性。正常的体细胞由于在细胞增殖过程中端粒DNA的丢失而具有有限的寿命。了解保护端粒免受快速缩短的机制对于预防过早衰老至关重要。研究发现，过度的端粒缩短可归因于C链填充缺陷、端粒复制应激和异常的端粒染色质结构。然而，这三种途径的机制知之甚少。最近，Ctc 1/Stn 1/Ten 1（CST）单链DNA结合复合物已被确定为保护端粒的新参与者。Ctc 1的突变导致一种称为Coats Plus的复杂疾病，来自Coats Plus患者的细胞显示短端粒。我们已经发现，CST复合物组分的缺乏会导致灾难性的端粒丢失，诱导人体细胞的早期细胞衰老和DNA损伤反应。因此，了解CST在防止端粒丢失中的功能是非常重要的。我们的数据表明，CST复合物是一种多功能的蛋白质，促进端粒DNA的有效复制，以及介导的C-链填充在染色体末端，以抵消过度端粒缩短。此外，我们的数据还表明，CST复合物可能参与调节端粒染色质结构，这是维持端粒长度的关键。本提案的目的是确定CST复合物在防止过度端粒丢失中的作用，以便制定治疗与端粒丢失相关的过早衰老的有效策略。设计实验以确定CST的翻译后修饰如何促进端粒DNA复制并精确调节C链填充（目的1和2）。此外，我们将确定复制叉停滞对端粒染色质组织的影响（目的3）。这将通过质谱、荧光DNA纤维分析和分子/生物化学方法的整合来实现。完成拟议的研究预计将大大增加我们对端粒维护的理解。这可能为研究端粒的维持和促进抗衰老治疗的发展带来新的方向。

DESCRIPTION (provided by applicant): Telomeres are the physical ends of chromosomes that protect genome stability by preventing chromosomes from being damaged. Normal somatic cells have limited lifespan due to the loss of telomere DNA during cell proliferation. Understanding the mechanisms that protect telomeres from rapid shortening is vital for preventing premature aging. Studies have discovered that excessive telomere shortening can be attributed to defective C-strand fill-in, telomere replication stress, and aberrant telomere chromatin structure. However, the mechanisms underlying these three pathways are poorly understood. Recently, the Ctc1/Stn1/Ten1 (CST) single-stranded DNA binding complex has been identified as a new player in protecting telomeres. Mutations in Ctc1 cause a complex disease known as Coats Plus, and cells derived from Coats Plus patients display short telomeres. We have found that deficiency in components of the CST complex causes catastrophic telomere loss, inducing early cellular senescence and DNA damage response in human somatic cells. Therefore, it is important to understand the functions of CST in preventing telomere loss. Our data suggest that the CST complex is a multifunctional protein that promotes efficient replication of telomeric DNA as well as mediates C-strand fill-in at chromosome ends to counteract excessive telomere shortening. In addition, our data also suggest that the CST complex may be involved in regulating telomeric chromatin structure, which is critical for maintaining telomere length. The objective of this proposal is to determine the roles of the CST complex in preventing excessive telomere loss, so that effective strategies for treating premature aging associated with telomere loss can be developed. Experiments are designed to determine how post-translational modification of CST facilitates telomeric DNA replication and precisely regulates C-strand fill- in (Aims 1 and 2). In addition, we will determine the effect of replication fork stalling on telomere chromatin organization (Aim 3). This will be accomplished by integration of mass spectrometry, fluorescent DNA fiber analysis, and molecular/biochemical methods. Completion of the proposed research is expected to substantially add to our understanding of telomere maintenance. It may lead to new directions in studying telomere maintenance and facilitating the development of anti-aging therapy.