细胞内G4结构形成对端粒DNA复制影响的研究

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。

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.