De novo telomere formation in wild type and mutant Arabidopsis

描述（由申请人提供）：端粒由富含G的串联重复序列及其相关蛋白组成。端粒蛋白调节端粒DNA束的长度并保护末端免受双链断裂（DSB）修复机制的影响。适当的端粒长度是通过缩短和延长端粒的过程之间的平衡来维持的。端粒的延伸主要是由端粒酶完成的。由于端粒酶可以将端粒重复序列添加到DSB中，因此有可能通过从头端粒形成（DNTF）来“修复”断裂的染色体。然而，在大多数情况下，其他DNA损伤修复机制占主导地位，没有观察到DNTF。维持端粒的完整性对于防止染色体末端融合至关重要，但染色体断裂处的DNTF可能导致缺失和更复杂的重排。在许多癌细胞中，染色体缺失和其他重排涉及DNTF。虽然DNTF所需的因素是未知的，端粒重复序列的存在增加了DNTF在附近的DSB的可能性。本研究将以模式生物拟南芥为材料，研究DNTF的作用过程，并确定影响其效率的因素。我将追求四个目标，利用遗传易处理性和广泛的突变体收集在拟南芥。前两个目标将集中在开发一个系统，以产生和检测拟南芥细胞中的DNTF事件。对于目标1，我将用含有端粒重复序列的构建体转化细胞，并测定DNTF导致的染色体截短。目的2：在植物中表达l-Scel限制性内切酶，在特定的转基因位点切割染色体。通过DNTF的DSB修复将通过远端标记基因的丢失来检测。在我的第三个目标中，我将测试不同大小、位置和方向的端粒重复序列对DNTF效率的影响。在最后的目标中，在先前的目标中开发的测定将用于比较不同遗传背景中DNTF的频率。所提出的实验测试端粒重复序列阵列在使染色体区域倾向于DNTF中的作用。此外，将测试DNTF的遗传要求。这些实验将提供深入了解内源性端粒是如何维持的，DNTF的过程，以及促进DNTF的因素。2.端粒，染色体的末端，是防止染色体重排所需的特殊结构。这种重排导致基因组不稳定，在癌细胞中很常见。这项研究着眼于这些结构是如何形成的，并测试它们的形成需要哪些基因。

DESCRIPTION (provided by applicant): Telomeres consist of G-rich tandem repeats and their associated protein. Telomere proteins regulate the length of the telomeric DNA tract and protect ends from double-strand break (DSB) repair mechanisms. Proper telomere length is maintained by equilibrium between processes that shorten the telomere tract and those that elongate it. Telomere extension is accomplished primarily by the telomerase enzyme. Because telomerase can add telomere repeats to DSBs, it is possible to "heal" broken chromosomes by de novo telomere formation (DNTF). However, in most cases other DNA damage repair mechanisms predominate and DNTF is not observed. Maintaining telomere integrity is critical to preventing chromosome end fusions, but DNTF at chromosome breaks could lead to deletions and more complex rearrangements. In many cancer cells, chromosome deletions and other rearrangements involve DNTF. Although the factors required for DNTF are unknown, the presence of telomere repeat arrays increases the likelihood of DNTF at nearby DSBs. This study will use the model organism Arabidopsis thaliana to study the process of DNTF and determine factors that influence its efficiency. I will pursue four aims that exploit the genetic tractability and extensive mutant collections available in Arabidopsis. The first two aims will focus on developing a system to produce and detect DNTF events in Arabidopsis cells. For Aim 1, I will transform cells with a construct containing a telomere repeat array and assay for chromosome truncation resulting from DNTF. For Aim 2, I will express the l-Scel restriction enzyme in planta to cleave chromosomes at a specific transgenic loci. DSB repair by DNTF will be detected by loss of distal marker genes. In my third aim, I will test the effects of telomere repeat arrays of various sizes, positions and orientations on the efficiency of DNTF. In the final aim, the assay developed in the previous aims will be used to compare the frequency of DNTF among different genetic backgrounds. The proposed experiments test the role of telomere repeat arrays in predisposing a chromosome region to DNTF. Furthermore, the genetic requirements of DNTF will be tested. These experiments will provide insight into how endogenous telomeres are maintained, the process of DNTF, and the factors that foster DNTF. 2. Telomeres, the ends of chromosomes, are specialized structures required to prevent chromosome rearrangement. Such rearrangements lead to genome instability and are common in cancer cells. This study looks at how these structures form and tests which genes are required for their formation.