Analysis of Telomerase-Independence Telomere Maintenance

描述（由申请方提供）：本提案的目的是表征非端粒酶依赖性端粒维持的机制。虽然端粒酶激活是维持端粒DNA的最常见机制，但一部分肿瘤和细胞系使用称为端粒替代延长（ALT）的端粒非依赖性机制来维持端粒DNA。在端粒酶缺陷的酵母菌株中，端粒稳定化是通过基于重组的机制实现的。同样，尽管分子机制尚未确定，但哺乳动物系统的证据与ALT涉及重组一致。基于酵母研究的数据，已经表明端粒重组需要失去最初定义端粒的加帽活性。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，将使用显性阴性和敲低方法鉴定参与DNA重组的ALT维持端粒所必需的因子。这些数据将表征ALT的分子要求，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的倾向相关的唯一遗传改变是p53状态。在其其他功能中，p53已被证明参与抑制重组。因此，p53在调节ALT中的作用将使用p53的温度敏感等位基因来表征。这些数据将是第一个说明一个因素的改变如何使细胞易患ALT的数据。端粒酶的抑制已经成为未来化疗药物的一个有吸引力的靶点。然而，ALT肿瘤对这些治疗是难治的。该提案将确定ALT激活所必需的结构变化和因子，从而确定化疗药物的新的潜在靶点。

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.