The Roles of the Ku Heterodimer in Yeast Telomere Function

基因组完整性受到DNA双链断裂（DSB）的威胁，如果不修复，可能导致 细胞周期停滞或死亡。因此，存在用于有效检测的复杂机制， 修复DSB产生的DNA末端。在天然染色体末端也会遇到DNA末端， 相反，必须保护其免受DSB修复活动，如非同源末端连接（NHEJ）， 来保持基因组的完整性这是通过专门的核蛋白结构来实现的， 端粒现在很清楚，许多响应DNA双链断裂的活动也在细胞中起作用。 正常的端粒结构、功能和维护。一种这样的蛋白质是Ku异源二聚体，其具有高亲和力， DNA末端结合复合物对NHEJ至关重要，尤其是端粒生物学的多个方面，如端粒末端结合复合物。 保护端粒免受异常修复活动的影响，调节端粒长度，以及形成一个 抑制性端粒染色质结构，导致附近基因的转录沉默，已知 端粒沉默。奇怪的是，Ku也是灾难性的端到端融合的主要效应器， 可能发生在功能失调的端粒上。Ku的NHEJ活性在野生型端粒中是如何被抑制的， 定义不好。PI和其他人以前的工作已经确定，Ku在 DSB与端粒，然而，在这些网站的作用机制尚未完全阐明。 最近，PI及其同事为Ku在DSB和端粒的功能开发了一个“双面”模型， 其中有一个向外的面，朝向DNA末端，介导NHEJ，和一个向内的面， 面，当与端粒结合时朝向端粒染色质，介导端粒功能。的 这项工作的总体目标是阐明Ku在端粒中活性的分子决定因素， 模式生物，酿酒酵母，从而扩展和测试双面模型。具体 目标1将a）进一步定义Ku的内表面，特别是关于Ku的端粒末端保护性质， 通过定点诱变; B）确定Ku的一种或多种端粒活性是否需要DNA末端 通过产生和表征DNA末端结合缺陷Ku蛋白来结合;和3）确定 通过分析Ku突变体的性质，与端粒末端相比，末端结合在保护断裂中的作用 仅由DNA结合核心组成。具体目标2将确定和表征蛋白质的相互作用 Ku在端粒末端保护或其他端粒功能中的作用。 特定目标3将通过识别因素进一步定义Ku修复特定外表的功能 与它所包含的NHEJ特异性表面<$-螺旋相互作用;这些将包括NHEJ因子以及 端粒因子可能抑制Ku介导的端粒NHEJ。因此，通过基因和 分子的方法，这一建议提供了作出实质性贡献，该领域的电流 了解Ku的功能，这可能会为Ku至关重要的人体细胞研究提供信息。

Genome integrity is threatened by DNA double strand breaks (DSBs), which, if left unrepaired, can lead to permanent cell cycle arrest or death. Consequently, complex mechanisms exist for the efficient detection and repair of DNA ends created by DSBs. DNA ends are also encountered at natural chromosome termini, which, conversely, must be protected from DSB repair activities, such as nonhomologous end joining (NHEJ), in order to preserve genome integrity. This is achieved through the specialized nucleoprotein structures known as telomeres. It is now clear that many of the activities that function in response to DNA DSBs also function in normal telomere structure, function, and maintenance. One such protein is the Ku heterodimer, a high affinity DNA end binding complex crucial for NHEJ and, notably, multiple aspects of telomere biology, such as the protection of telomeres from aberrant repair activities, the regulation of telomere length, and the formation of a repressive telomeric chromatin structure, which results in the transcriptional silencing of nearby genes, known as telomeric silencing. Paradoxically, Ku is also a principal effector of the catastrophic end-to-end fusions that can occur at dysfunctional telomeres. How Ku's NHEJ activity is inhibited at wild type telomeres remains poorly defined. Previous work by the PI and others has firmly established that Ku performs distinct activities at DSBs vs. telomeres, however the mechanisms of action at these sites have yet to be fully elucidated. Recently, the PI and co-workers have developed a `two-face' model for Ku's functions at DSBs and telomeres, in which there is an outward face, oriented toward the DNA terminus, which mediates NHEJ, and an inward face, oriented toward telomeric chromatin when bound to a telomere, which mediates telomeric functions. The overall goal of the proposed work is to elucidate the molecular determinants of Ku's activities at telomeres in the model organism, Saccharomyces cerevisiae, thereby expanding and testing the two-face model. Specific Aim 1 will a) further define Ku's inward face, particularly with respect to Ku's telomere end protection property, via site-directed mutagenesis; b) determine whether one or more of Ku's telomeric activities require DNA end binding by generating and characterizing DNA end binding defective Ku proteins; and 3) determine the role of end binding in protecting broken as compared to telomeric ends by analyzing the properties of Ku mutants consisting of solely the DNA binding core. Specific Aim 2 will identify and characterize proteins that interact with Ku in telomere end protection or other telomeric functions using genetic and biochemical approaches. Specific Aim 3 will further define the function at Ku's repair-specific outward face by identifying the factor(s) that interact with an NHEJ-specific surface ¿-helix it contains; these will include NHEJ-factors as well as telomeric factors that may inhibit Ku-mediated NHEJ at telomeres. Thus, through a combination of genetic and molecular approaches, this proposal offers to make a substantial contribution to the field's current understanding of the function of Ku, which may inform studies in human cells, where Ku is essential.