RecQ4重启端粒受阻复制叉机制的研究

Telomere DNA with reparative guanine-rich sequence were thought to be a physical blockade of the end chromosomes. DNA replication in telomere area is deemed to be difficult compared with the other parts of genome, which requires additional co-factors to facilitate the process. In the previous study, we have reported that the newly identified telomere binding protein complex CTC1-STN1-TEN1 could reinitiate the stalled telomere replication forks, through firing the dominate origins nearby. However, CST complex could only bind to the ssDNA and doesn't exhibit the properties of DNA polymerase, topoisomerase or helicase, which is required for the DNA replication re-initiation, suggesting there must be some other replication factors participating in the process. To further investigate the mechanism of replication re-initiation in telomere, we managed to identify the proteins which could interact with CST, and RECQ4 turns out to be one of these co-factors. RECQ4 is a member of RECQ helicase family, which could unwind double strand DNA and initiate the replication. Mutations in RECQ4 lead to specific features of Rothmans-Thomson syndrome, including spares hair, skeletal abnormalities, short life span and increased risk of cancer. Moreover, we identified that RECQ4 knockout could induce telomere replication disorders as well as stalled telomere replication forks. Based on these observations, we raised our proposal, that is "When replication fork stalling occurs, CST could locate to these regions via its ssDNA binding domain, subsequently RECQ4 could be recruited to the stalled forks through the interaction with CST, then initiates the Pre-initiation complex formation and restarts the stalled replication". We are going to further investigate the mechanism of RECQ4 in restarting the stalled replication fork restart here, it will not only broad our knowledge of telomere function of RECQ4 but also guide our current aging and cancer therapy which based on regulating telomere replication.

端粒DNA序列高度重复，复制较为困难，通常需要额外的蛋白因子进行辅助。CST蛋白能够激活受阻复制叉、重启受阻端粒复制。然而其并不具备起始DNA复制所必需的酶活性，重启复制的机制尚未明确。我们发现解旋酶RECQ4可以与CST发生相互作用，同时解旋高级结构并与MCM10协同作用起始DNA合成，我们的结果还进一步证实其敲除能够导致端粒复制叉的停滞并引发复制性损伤。据此我们提出以下论点，“端粒复制受阻时，CST与所产生的单链端粒DNA相互结合，随后RECQ4通过与CST的相互作用结合到受阻复制叉，并与MCM10一起在相邻的休眠复制起始位点，共同组装复制前体，重启受阻复制位点”。本课题中我们将系统的研究RECQ4重启端粒受阻复制叉的具体机制，该研究将开拓对RECQ4端粒功能的探索，并为进一步明确端粒DNA复制的分子机制奠定基础，同时为以调控端粒复制为手段的肿瘤或衰老治疗提供方案。

在哺乳动物中，端粒是由TTAGGG的重复序列及其结合蛋白共同组成。端粒的主要是功能是维持染色体完整性、防止染色体融合及保持基因组稳定性。由于端粒DNA高度重复以及富含鸟嘌呤的特点，其极易形成高级结构，比如T-loop，D-loop以及G-quadruplex（G4）等。由于该类结构的存在，DNA双链不能作为DNA复制模板。因此为使端粒得以正常复制，此类高级结构需要被打开。我们前期发现端粒结合复合体CST也具备结合G4的功能，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致停滞；另外，CST可以与G4结合，定位在受阻复制叉上使其重新启动。但是，CST结合G4重启受阻复制叉的机制还不明确，我们猜想可能有其他蛋白与CST作用进而发挥功能。我们利用质谱分析与CST相互作用的蛋白，发现RECQ4解旋酶与CST发生显著相互作用。RECQ4是RECQ解旋酶家族成员，在DNA复制过程中高级结构的解旋打开发挥着重要作用。本项目中，我们分别面探讨了RECQ4在基因组和端粒上的生物学功能，阐明了其在端粒复制及端粒DNA损伤中的作用，并最终明确了RECQ4与CST相互作用重启端粒受阻复制叉的分子机制，为更加全面了解肿瘤细胞中端粒DNA复制的机制奠定基础，给以抑制端粒复制为手段的肿瘤治疗提供新的靶标和方案。