Role of human CST in preventing telomere loss

描述(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的稳定性。由于端粒DNA在细胞增殖过程中的丢失，正常体细胞的寿命有限。了解保护端粒免受快速缩短的机制对于防止过早衰老至关重要。研究发现，端粒过度缩短可归因于C链填充缺陷、端粒复制压力和端粒染色质结构异常。然而，这三条途径背后的机制却知之甚少。最近，Ctc1/Stn1/Ten1(CST)单链DNA结合复合体被确定为保护端粒的新角色。Ctc1的突变会导致一种名为Coats Plus的复杂疾病，来自Coats Plus患者的细胞显示出较短的端粒。我们发现，CST复合体成分的缺乏会导致灾难性的端粒丢失，导致细胞提前衰老和人体细胞的DNA损伤反应。因此，了解CST在预防端粒丢失中的作用是非常重要的。我们的数据表明，CST复合体是一种多功能蛋白质，它促进端粒DNA的有效复制，并介导染色体末端的C链填充，以抵消过度的端粒缩短。此外，我们的数据还表明，CST复合体可能参与调节端粒染色质结构，这是维持端粒长度的关键。这项建议的目的是确定CST复合体在防止过度端粒丢失中的作用，以便开发有效的策略来治疗与端粒丢失相关的过早衰老。实验旨在确定CST的翻译后修饰如何促进端粒DNA复制和精确调节C链填充(目标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(目标3)。这将通过集成质谱学、荧光DNA纤维分析和分子/生化方法来实现。这项拟议研究的完成有望大大增加我们对端粒维持的理解。这可能为研究端粒维护和促进抗衰老治疗的发展提供新的方向。

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.