Roles of the mammalian CST complex in DNA replication and chromosome cohesion

描述（由申请人提供）：我们目前对DNA复制如何通过自然发生的屏障进行以及姐妹染色单体内聚（SCC）如何在DNA复制过程中建立的理解存在根本差距。在基因组的复制过程中，复制体复合物会遇到各种各样的障碍。这些障碍包括非自然障碍和自然障碍。非自然障碍包括DNA损伤和双链断裂。天然障碍包括重复DNA、DNA结合蛋白和RNA转录位点。根据人类基因组的大小，预计在整个s期过程中，复制体复合体会在自然障碍中多次停滞。由于这些天然障碍不需要修复，细胞已经进化出机制来防止这些障碍引起DNA损伤反应和细胞周期停滞。然而，这是如何发生的仍然知之甚少。该项目的长期目标是阐明DNA复制如何通过自然染色体屏障进行，例如重复DNA序列和DNA结合蛋白。该职业发展奖的目标有两个方面：1)完成研究计划的具体目标，即确定CTC1-STN1-TEN1 （CST）复合物在DNA复制和SCC中的非端粒作用；2)在该奖项的K99阶段，通过学习新技术、开发独立项目、获得教学经验和接受指导指导来获得职业发展。K99阶段将在辛辛那提大学的Carolyn Price博士的指导和指导下进行，并得到dr。Paul Chastain, David Kaufman， Prasad Jallepalli和Birgit Ehmer博士。我们基因组中阻碍复制的天然障碍包括难以复制的DNA区域，如端粒、脆弱位点、三核苷酸重复和着丝粒DNA。在这些区域，复制体必须在停止后重新启动。然而，DNA合成是如何在这些位点重新启动的，仍然没有得到很好的描述。SCC是在DNA复制过程中建立的，并被认为与复制体的传代同时发生。令人惊讶的是，我的初步研究结果表明，新发现的端粒相关CST复合物不仅在端粒上起作用，而且在DNA复制重启和SCC中都起作用。有趣的是，其他几种DNA复制蛋白的消耗导致SCC缺陷和DNA复制重启，这表明这两个过程之间存在联系。CST的两个组分CTC1和STN1最初被鉴定为DNA聚合酶α -引物酶（pol α）辅助因子，刺激pol α结合和引物酶活性。CST还能结合ssDNA，其结构类似于复制/修复因子复制蛋白A （RPA）。总之，这些发现表明CST与pol α的相互作用对其非端粒功能很重要。该建议的中心假设是，CST通过促进快速复制重启和难以到达的位点的SCC来防止基因组不稳定

DESCRIPTION (provided by applicant): There is a fundamental gap in our current understanding of how DNA replication proceeds through naturally occurring barriers and how sister chromatid cohesion (SCC) is established during DNA replication. During replication of the genome, the replisome complexes encounter a variety of barriers. These include unnatural and natural impediments. Unnatural impediments include DNA lesions and double-strand breaks. Natural impediments include repetitive DNA, DNA-bound proteins and sites of RNA transcription. Based on the size of the human genome, replisome complexes are predicted to stall many times at natural impediments throughout the course of S-phase. Since these natural impediments do not require repair, the cell has evolved mechanisms to prevent these impediments from causing a DNA damage response and cell cycle arrest. However, how this occurs remains poorly understood. The long-term goal of this project is to elucidate how DNA replication proceeds through natural chromosome barriers, such as repetitive DNA sequences and DNA-bound proteins. The objective of this career development award is two-fold: 1) complete the specific aims of the research proposal, which are to determine the non-telomere roles of the CTC1-STN1-TEN1 (CST) complex in DNA replication and SCC, and 2) receive career development through learning new techniques, developing an independent project, gaining teaching experience and receiving guided mentoring in the K99 phase of this award. The K99 phase will occur under the mentorship and guidance of Dr. Carolyn Price at the University of Cincinnati with additional support from Drs. Paul Chastain, David Kaufman, Prasad Jallepalli and Dr. Birgit Ehmer. Natural impediments in our genome that stall replication include difficult-to-replicate DNA regions such as telomeres, fragile sites, trinucleotide repeats and centromeric DNA. At these regions, the replisome must be restarted after stalling. However, how DNA synthesis is reinitiated at these sites remains poorly characterized. SCC is established during DNA replication and proposed to occur concurrent with passage of the replisome. Surprisingly, my preliminary findings suggest that the newly discovered, telomere-associated CST complex not only functions at the telomere but also in both DNA replication restart and SCC. Interestingly, depletion of several other DNA replication proteins leads to defects in SCC and DNA replication restart, suggesting a link between these two processes. Two components of CST, CTC1 and STN1, were originally identified as DNA polymerase alpha- primase (pol alpha) accessory factors, which stimulate pol alpha binding and primase activities. CST also binds ssDNA and is structurally similar to the replication/repair factor replication protein A (RPA). Together, these findings suggest that CST interactions with pol alpha are important for its non-telomere functions. The central hypothesis of this proposal is that CST prevents genome instability by promoting rapid replication restart and SCC at sites of difficult-to replicate DNA, such as telomeres and fragile sites. The proposed research will address this hypothesis through three specific aims: 1) To determine the mechanism by which CST facilitates replication restart after fork stalling; 2) To elucidate the role of CST in sister chroatid cohesion and mitotic progression; 3) To identify CST interactions with replication restart and sister chromatid cohesion factors. In the first aim, the role of CST in replication restart will be investigated by analyzing restart at both the cellular and molecular level in CST-depleted cell lines, determining whether CST is localized to sites of fork stalling, analyzing replication fork stalling in CST-depleted cell lines at sites of difficult-to-replicate DNA and characterizing CST ssDNA binding activity. To perform these experiments, I will receive training in DNA fiber analysis from Dr. Paul Chastain at the University of North Carolina, employ a new protocol for isolating DNA at stalled replication forks and utilize my biochemical and cell biology training. In the second aim, the role of CST in SCC will be assessed by first determining the timing of cohesion loss and whether defects in mitotic progression arise from SCC loss in CST-depleted cells. These studies will require me to learn live-cell imaging and new cell biology techniques. For these studies, I will be collaborating with Dr. Prasad Jallepalli, associate member and laboratory head at the Memorial Sloan-Kettering Cancer Center and an expert in chromosome cohesion and mitosis. The third aim will use a multi-pronged approach to determine CST interacting partners. These studies will include hypothesis-driven experiments to identify CST interactions with proteins involved in DNA replication restart and SCC. CST pull-down followed by mass spectrometry will be used as an unbiased approach to gain insight into CST function through the identification of novel interacting peptides. This proposed work is innovative because: 1) it addresses the unexpected non-telomere functions of CST; 2) it investigates novel mechanisms for the reinitiation of DNA synthesis after fork stalling at natural impediments; 3) it combines a variety of new and well-established techniques to investigate the central hypothesis. The work is significant because it will reveal some of the underlying mechanisms of chromosome instability. Each time a cell divides its DNA must be properly replicated and SCC maintained to ensure proper chromosome segregation to the daughter cells. Defects in either DNA replication or chromosome cohesion lead to phenotypes associated with cancer initiation, such as translocations, deletions, chromosome fusions, gene duplication and aneuploidy. Several genetic disorders, termed cohesionopathies, are also associated with SCC loss and chromosome breakage. Furthermore, mutations in CTC1 were recently shown to underlie a rare autosomal recessive disorder, Coats plus. The completion of these studies will advance our understanding of these cellular processes and provide new targets for prevention and treatment of these diseases.