Analysis of Telomerase-Independence Telomere Maintenance

描述(由申请人提供)：本提案的目的是描述端粒酶非依赖性端粒维持的机制。虽然端粒酶激活是维持端粒DNA最常见的机制，但一些肿瘤和细胞系使用一种端粒酶不依赖的机制，称为端粒交替延长(ALT)，以维持端粒DNA。在端粒酶缺乏的酵母菌株中，端粒稳定是通过基于重组的机制实现的。同样，尽管分子机制尚未建立，但来自哺乳动物系统的证据与涉及重组的ALT是一致的。根据酵母研究的数据，有人认为端粒重组需要失去最初定义端粒的封顶活性。为了在哺乳动物细胞中验证这一假设，将使用分子生物学方法和电子显微镜来确定ALT端粒的结构。阐明ALT端粒的结构将有助于深入了解ALT的分子机制。其次，参与DNA重组的因素是ALT维持端粒所必需的，将使用显性负性和敲除方法进行鉴定。这些数据将表征ALT的分子需求，并将区分可能的基于重组的机制。哺乳动物细胞中与激活ALT的易感性有关的唯一基因改变是P53状态。在它的其他功能中，P53被证明参与了重组的抑制。因此，将利用P53的温度敏感等位基因来表征P53在调节ALT中的作用。这些数据将是第一个说明一个因子的改变可能如何使细胞容易发生改变的数据。抑制端粒酶活性已成为未来化疗药物的研究热点。然而，ALT肿瘤对这些治疗方法是难治的。这项建议将确定ALT激活所必需的结构变化和因素，从而确定新的潜在化疗药物靶点。

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize the mechanism of telomerase-independent telomere maintenance. Although telomerase activation is the most common mechanism for maintenance of telomeric DNA, a subset of tumors and cell lines use a telomerase-independent mechanism, termed Alternative Lengthening of Telomeres (ALT), to sustain telomeric DNA. In telomerase-deficient yeast strains, telomere stabilization is achieved through recombination-based mechanisms. Similarly, although the molecular mechanism has yet to be established, the evidence from mammalian systems is consistent with ALT involving recombination. Based on data from yeast studies, it has been suggested that telomere recombination requires loss of the capping activity by which telomeres were originally defined. To test this hypothesis in mammalian cells, the structure of ALT telomeres will be determined using molecular biological methods and electron microscopy. Clarifying the structure of ALT telomeres will provide insight into the molecular mechanism of ALT. Secondly, factors involved in DNA recombination that are essential for telomere maintenance by ALT will be identified using dominant negative and knockdown approaches. These data will characterize the molecular requirements for ALT and will differentiate between possible recombination-based mechanisms. The only genetic alteration in mammalian cells linked to a predisposition to activate ALT is p53 status. Among its other functions, p53 has been shown to be involved in suppression of recombination. Thus, the role of p53 in regulating ALT will be characterized using temperature sensitive alleles of p53. These data will be the first that illustrate how alterations in a factor might predispose cells to ALT. Inhibition of telomerase has become an attractive target for future chemotherapeutic agents. However, ALT tumors would be refractory to these treatments. This proposal will identify structural changes and factors essential for activation of ALT and thus identify new potential targets for chemotherapeutic agents.