Roles of CST, a novel telomere associated complex, at the DNA replication fork

描述(申请人提供)：本项目的总体目标是通过难以复制的DNA位置了解DNA复制的机制。这些将包括被预测形成DNA二级结构的部位，如端粒和脆性部位。端粒由重复的富含G-C的DNA组成，长期以来一直被认为是复制叉机械的天然障碍。脆性部位表现为染色体DNA在复制应激条件下出现的缺口或断裂。众所周知，端粒和脆性部位会导致基因组不稳定，如果维护不当，可能会导致癌症发生。CTC1-STN1-TEN1(CST)是最近在植物和脊椎动物中发现的一种新的蛋白质复合体。STN1和TEN1是酵母蛋白的同系物，参与端粒维持和末端保护。在人类中，CST被证明定位于端粒。人类CST亚基的缺失导致基因组不稳定的特征，如3H2 AX焦点和染色质桥增加，以及端粒异常，如G-突出增加和端粒信号丢失。几个实验室的结果表明，CST既具有端粒功能，也具有非端粒功能。重要的是，CST被证明与DNA聚合酶1-Primase(Pol1)相互作用，启动DNA合成。这项研究的初步结果表明，CST在端粒复制和复制分叉重新启动中发挥作用。我们建议CST招募DNA Pol1在难以复制的DNA位点重新启动DNA合成，这可能会阻碍DNA复制。在目标1中，将研究CST在端粒复制中的作用。然后将扩大研究范围，以确定是否需要CST来维持染色体脆性部位。最后，将测量CST和RecQ解旋酶WRN和BLm之间的相互作用在多大程度上需要维持端粒和脆性位点的稳定性。在目标2中，将研究CST促进复制分叉进展的机制。叉子失速后的叉子重新启动和单链DNA水平将在CST耗尽的细胞系中进行测量。然后将探索CST与复制分叉组件和分叉重新启动因子的相互作用。总之，这些研究将提供对CST如何通过难以复制的DNA来帮助维持基因组稳定性的理解。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand the mechanism of DNA replication through difficult-to-replicate sites of DNA. These would include sites predicted to form DNA secondary structure such as telomeres and fragile sites. Telomeres consist of repetitive, G-C rich DNA and have long been considered natural impediments to the replication fork machinery. Fragile sites are expressed as gaps or breaks in chromosomal DNA that occur during conditions of replication stress. Telomeres and fragile sites are known to cause genomic instability and can lead to carcinogenesis when not properly maintained. CTC1-STN1-TEN1 (CST) is a novel protein complex, which was recently discovered in plants and vertebrates. STN1 and TEN1 are homologues to yeast proteins, which are involved in telomere maintenance and end-protection. In humans, CST was shown to localize to telomeres. Depletion of human CST subunits results in hallmarks of genomic instability, i.e. increased 3H2AX foci and chromatin bridges, as well as telomere abnormalities such as increased G-overhangs and telomere signal loss. Results from several labs suggest that CST has both telomeric and non- telomeric functions. Importantly, CST was shown to interact with DNA polymerase 1-primase (pol 1), which initiates DNA synthesis. Preliminary results presented in this proposal suggest that CST functions in telomere replication and replication fork reinitiation. We propose that CST recruits DNA pol 1 to reinitiate DNA synthesis at sites of dificult-to-replicate DNA, which may stall DNA replication. In aim 1, the role of CST in telomere replication will be investigated. Studies will then be extended to determine whether CST is required to maintain chromosomal fragile sites. Finally, the extent to which interactions between CST and the RecQ helicases, WRN and BLM, are required to maintain telomere and fragile site stability will be measured. In aim 2, the mechanism by which CST promotes replication fork progression will be studied. Fork reinitiation after fork stalling and the levels of single-stranded DNA will be measured in CST-depleted cell lines. CST interactions with replication fork components and fork reinitiation factors will then be explored. Together, these studies will provide an understanding of how CST helps maintain genomic stability through difficult-to-replicate DNA.