Telomere structure and function in Arabidopsis

 描述(申请人提供)：端粒通过区分自然的染色体末端和DNA损伤(末端保护)和通过端粒酶促进DNA末端的复制(端粒维护)来稳定基因组。这些功能是通过动态分子开关执行的，该开关将染色体末端从完全保护(端粒酶不可延伸)构象转换为开放(端粒酶可延伸)状态。这种开关的分子基础尚不清楚，但必须严格控制，因为末端暴露不当或未能完全复制会导致干细胞疾病和癌症。在这一新的应用中，我们利用真核细胞拟南芥模型来揭示端粒酶和CST(CTC1/STN1/TEN1)端粒蛋白复合体之间的相互作用。该应用建立在前一个资助期的重要发现的基础上，这些发现包括：1)与与染色体末端保护有关的蛋白质结合伙伴的替代端粒酶RNP复合体；2)正向调节端粒酶的端粒酶辅助因子POT1a和负向调节端粒酶的CST的核心成分TEN1之间的动态切换；以及3)非规范的端粒酶RNA(TER2)，它负向调节端粒酶的活性以响应DNA损伤。这一应用的中心假设是端粒酶组件与CST一起协调末端保护和复制复合体的组装和动态交换，以促进端粒的稳定性。这一假设将通过两个具体的目标来检验，这两个目标结合使用了生化、遗传学和细胞生物学的方法。目标1将研究钝端端粒是如何稳定的。这项工作将集中在Ku，POT1b和POT1c在端粒保护中的作用，并将检验一种加工形式的TER2，TER2s，作为RNA支架覆盖钝端端粒的假设。目的2定义CST、端粒酶辅助蛋白和TER2调控RNA之间的动态相互作用，以阐明这些因素如何控制端粒酶的端粒合成。目的2研究TEN1和TER2在端粒酶介导的端粒延长中的作用。这些研究的影响是，他们将建立端粒保护和端粒酶调节的新范式，并深入了解长非编码RNA在促进基因组完整性方面的新角色。从更广泛的意义上讲，这些结果将解释如何利用端粒成分的快速进化来实现端粒稳态的常见机制以及端粒调节的新模式。由于在拟南芥中发现的复杂的端粒酶调节机制可能是保守的，这些目的的完成将提供关于端粒酶和CST相关人类疾病的重要新信息。

 DESCRIPTION (provided by applicant): Telomeres stabilize the genome by distinguishing natural chromosome ends from DNA damage (end protection) and by facilitating replication of terminal DNA tracts via telomerase (telomere maintenance). These functions are executed through a dynamic molecular switch that converts the chromosome terminus from a fully protected (telomerase non-extendable) conformation into an open (telomerase-extendable) state. The molecular basis of the switch is unknown, but it must be strictly controlled as inappropriate exposure of the terminus or failure to fully replicate it leads to stem cell disease and cancer. In this renewal application we exploit the model eukaryote Arabidopsis to reveal new insight into the interplay between telomerase and the CST (CTC1/STN1/TEN1) telomere protein complex. The application builds on important discoveries from the previous funding period that include: 1) an alternative telomerase RNP complex with protein binding partners implicated in chromosome end protection; 2) a dynamic switch between a telomerase accessory factor POT1a, which positively regulates telomerase, and TEN1 a core component of CST, which negatively regulates telomerase; and 3) a non-canonical telomerase RNA (TER2) that negatively regulates telomerase enzyme activity in response to DNA damage. The central hypothesis of this application is that telomerase components together with CST orchestrate the assembly and dynamic exchange of end protection and replication complexes to promote telomere stability. This hypothesis will be tested through two specific Aims that employ a combination of biochemical, genetic and cell biological approaches. Aim 1 will investigate how blunt-end telomeres are stabilized. This work will focus on the roles of Ku, POT1b and POT1c in telomere protection, and will test the hypothesis that a processed form of TER2, TER2s, serves as an RNA scaffold to cap blunt-end telomeres. Aim 2 will define the dynamic interactions between CST, telomerase accessory proteins and the TER2 regulatory RNA to elucidate how these factors control telomere synthesis by telomerase. Aim 2 focuses on the roles of TEN1 and TER2 in terminating telomerase-mediated telomere elongation. The impact of these studies is that they will establish new paradigms for telomere protection and telomerase regulation, and yield insight into the emerging roles of long noncoding RNA in promoting genome integrity. In a broader sense, the results will explain how rapid evolution of telomere components can be used to achieve common mechanisms of telomere homeostasis as well as new modes of telomere regulation. Since it is likely that the sophisticated mechanisms of telomerase regulation discovered in Arabidopsis are conserved, completion of these aims will provide important new information about telomerase and CST related human disease.