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，可能会导致 永久性细胞周期停滞或死亡。因此，存在用于有效检测和 双链断裂产生的DNA末端的修复。DNA末端也在自然的染色体末端遇到， 相反，必须防止DSB修复活动，如非同源末端连接(NHEJ)，以便 以保持基因组的完整性。这是通过特殊的核蛋白结构实现的，称为 端粒。现在很清楚的是，许多对DNADSB起作用的活动也在 正常的端粒结构、功能和维持。其中一种蛋白质是Ku异源二聚体，一种高亲和力的蛋白质 DNA末端结合复合体对NHEJ以及端粒生物学的多个方面至关重要，例如端粒 保护端粒不受异常修复活动的影响，调节端粒长度，形成 抑制性端粒染色质结构，导致附近基因转录沉默，已知 作为端粒沉默。矛盾的是，Ku也是灾难性的端到端融合的主要执行者 可能发生在功能失调的端粒。Ku的NHEJ活性是如何在野生型端粒上被抑制的仍然存在 定义不明确。PI和其他人之前的工作已经牢固地确立了Ku在 DSB与端粒的比较，然而这些部位的作用机制还没有完全阐明。 最近，Pi和他的同事们开发了Ku在DSB和端粒上的功能的“两面”模型， 其中有一个朝向DNA末端的向外的面，它介导着NHEJ，以及一个向内的 与端粒结合时朝向端粒染色质的脸，它调节端粒的功能。这个 这项拟议工作的总体目标是阐明Ku在端粒活性的分子决定因素。 模型生物，酿酒酵母，从而扩展和测试双面模型。特定的 目标1)将进一步定义Ku的内向表面，特别是关于Ku的端粒末端保护特性， 通过定点突变；b)确定Ku的一个或多个端粒活性是否需要DNA末端 通过产生和鉴定DNA末端结合缺陷Ku蛋白来结合；以及3)决定 通过分析Ku突变体的性质比较末端结合保护断裂末端与端粒末端的差异 仅由DNA结合核心组成。特殊目标2将识别和表征相互作用的蛋白质 Ku具有端粒末端保护或其他端粒功能，使用遗传和生化方法。 具体目标3将通过确定因素进一步定义辜朝明修复特定外在脸部的功能(S) 与其包含的NHEJ特定表面-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，通过基因和基因的结合 分子方法，这项提议提供了对该领域当前的实质性贡献 了解Ku的功能，这可能会为人类细胞的研究提供信息，而Ku是必不可少的。

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.