细胞内G4结构形成对端粒DNA复制影响的研究

G4 are secondary structures that may form within guanine-rich telomere DNA sequences. They were thought to contribute to cap telomeres and pose a physical blockade for the access of telomerase to the chromosomes ends. Furthermore, the in vitro study showed G4 structure could also cause replication stress and inhibit DNA replication. However, the in vivo functions for G4 structure haven't been cleared, due to the lack of suitable techniques. In this project, we are going to investigate its telomere functions using several techniques recently developed in our lab; meanwhile, the numbers of G4 formed in cells will be quatified using the newest developed G4 antibody directly. Our current data suggested that, the formation of G4 structure could lead to replication fork stalling and telomere dysfunction induced foci in the cells. Moreover, we identified that the G4 structure could be bound and unfolded by（CTC1-STN1-TEN1）CST complex. Since, the CST complex was reported to facilite DNA replicaiton restart after replication fork stalling, we proposed that G4 structure has two independent functions during telomere replication: first, the formation of G4 could cause telomere replication stress and lead to replication fork stalling, result in cell apoptosis or senescence; 2nd,G4 structure can be recognized by CST complex and DNA polymerase α could be recruited to the stalled replication forks throught its interaction with CST, result in stalled replication fork restart. In this study, the investigation of the functions of G4 structure during telomere replication, guide our current aging and cancer therapy which based on regulating telomere replication.

端粒DNA能够折叠成G4高级结构，体外研究发现G4的形成能抑制端粒酶对端粒的识别和延伸，同时能够阻碍端粒DNA复制的正常进行。然而由于缺乏有效的手段，体内G4的端粒生物学功能尚未明确，本课题中我们拟利用新研发的G4抗体观察该结构在体内的形成，结合我们独立创新的技术研究其对端粒复制的影响。初步实验数据表明，体内过量G4的形成可以造成端粒复制叉的停滞并引起端粒损伤；同时该结构能与CTC1-STN1-TEN1（CST）复合体相互结合，CST在体内的一个主要功能是与DNA聚合酶α作用并重启受阻DNA的复制。因此我们认为"G4结构在体内的形成导致复制叉的停滞并引发端粒损伤；而CST能够与G4相互识别，打开该结构并招募复制体蛋白以重启受阻复制叉"。本课题中我们将系统的研究G4形成对端粒复制的影响，该研究将为以调控端粒复制为手段的肿瘤和衰老治疗提供新的靶标和方案。

研究背景：端粒位于染色体末端，具有稳定染色体、防止染色体融合、保护基因组的作用。由于其高度重复的特点，端粒DNA能够形成高级结构G-quadruplex （G4）影响其正常复制。其复制的失误可导致端粒功能的紊乱或端粒的丢失，进而引发细胞的衰老或凋亡。对 G4 结构以及 其生物学功能的研究，将有助于我们更加全面的了解癌细胞中端粒 DNA复制的机制，并为以抑制端粒复制为手段的肿瘤治疗提供新的靶标和方案。.主要内容和结果：我们研究发现，G4结构的形成能够阻止端粒区复制叉正常移动，进而可能造成DNA损伤修复通路ATR的激活。而ATR激活所导致的细胞内单链DNA的累积可能进一步诱发端粒区形成G4结构，最终导致端粒功能紊乱并引起全基因组DNA复制失调。端粒作为感受DNA复制压力最敏感的区域，其损伤极易引发细胞的衰老或凋亡。为保证DNA复制的顺利进行，形成的G4结构需要被打开，受阻复制叉需要得到重启。我们发现的端粒结合复合体CST具备结合G4的能力，其缺失促成体内G4结构的积累，并导致端粒区复制叉移动变缓以至停滞。在进一步的研究中，我们利用单分子荧光技术对端粒区的DNA复制进行研究，证实CST可以与G4结合，定位到端粒受阻复制叉上，通过激活邻近的休眠复制起始位点使受阻复制叉重新启始。.关键数据：1）复制压力造成ssDNA 累积，诱导G4 结构形成.进而引发端粒复制性紊乱端粒丢失以及端粒损伤；2）G4诱导产生的端粒功能障碍可被端粒酶所恢复; 3）端粒结合蛋白可以直接打开G4结构; 4）CST蛋白介导G4结构打开，调控端粒DNA后随链的复制。.科学意义：然而由于缺乏在体内直接观察 G4 结构的技术方 法，迄今为止没有任何直接的证据明确该结构在体内的生物学功能。而本项目的最大突破，就在于我们借助G4抗体首次在细胞内明确 G4结构的端粒生物学功能。同时我们发现端粒结合复合体CST可打开G4结构，辅助端粒后随链合成。