The Roles of the Ku Heterodimer in Yeast Telomere Function

描述（由申请人提供）：基因组完整性受到DNA双链断裂（DSB）的威胁，如果不修复，可能导致永久性细胞周期停滞或死亡。因此，存在复杂的机制来有效检测和修复由DSB产生的DNA末端。在天然染色体末端也会遇到DNA末端，相反，为了保持基因组的完整性，必须保护其免受DSB修复活动，如非同源末端连接（NHEJ）。这是通过被称为端粒的特殊核蛋白结构实现的。现在很清楚，许多响应DNA双链断裂的活动也在正常的端粒结构、功能和维护中发挥作用。一种这样的蛋白质是Ku异二聚体，一种对NHEJ至关重要的高亲和力DNA末端结合复合物，并且值得注意的是，端粒生物学的多个方面，例如保护端粒免受异常修复活性的影响，调节端粒长度，以及形成抑制性端粒染色质结构，其导致附近基因的转录沉默，称为端粒沉默。奇怪的是，Ku也是可能发生在功能失调的端粒处的灾难性端对端融合的主要效应物。Ku的NHEJ活性如何在野生型端粒处被抑制仍然不清楚。PI和其他人先前的工作已经确定Ku在DSB与端粒上进行不同的活动，但是这些位点的作用机制尚未完全阐明。最近，PI和同事们已经开发了一个“双面”模型Ku的功能在DSB和端粒，其中有一个向外的脸，面向DNA末端，介导NHEJ，和向内的脸，面向端粒染色质时，绑定到端粒，介导端粒的功能。拟议的工作的总体目标是阐明Ku的活动在端粒的模式生物，酿酒酵母的分子决定因素，从而扩大和测试的双面模型。具体目标1将a）通过定点诱变进一步限定Ku的内向面，特别是关于Ku的端粒末端保护性质; B）通过产生和表征DNA末端结合缺陷Ku蛋白来确定Ku的端粒活性中的一种或多种是否需要DNA末端结合;和3）通过分析仅由DNA结合核心组成的Ku突变体的性质，确定与端粒末端相比，末端结合在保护断裂中的作用。具体目标2将确定和表征蛋白质相互作用与Ku在端粒末端保护或其他端粒功能，使用遗传和生物化学方法。具体目标3将通过鉴定与其所含的NHEJ特异性表面1-螺旋相互作用的因子来进一步定义Ku修复特异性外表面的功能;这些因子将包括NHEJ因子以及可能在端粒抑制Ku介导的NHEJ的端粒因子。因此，通过遗传和分子方法的结合，该提案提供了对该领域目前对Ku功能的理解做出实质性贡献，这可能会为Ku至关重要的人类细胞研究提供信息。

DESCRIPTION (provided by applicant): 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 1-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.