Mechanisms of formation of terminal deletions

描述（申请人提供）：端粒的主要功能是保护染色体末端，防止染色体融合。染色体融合可通过断裂/融合/桥（B/F/B）循环导致染色体不稳定性，当染色体在每次细胞分裂中重复断裂和融合时发生。通过在断裂染色体的末端添加端粒，可以阻止或终止B/F/B循环，这导致末端缺失的形成。在四膜虫和酵母中，端粒被端粒酶添加到断裂染色体的末端，称为染色体愈合。与端粒丢失和B/F/B周期相关的染色体重排已在多种人类遗传性疾病中发现，并且被认为在与癌症相关的染色体不稳定性中发挥重要作用。我们已经建立了一种利用位于端粒附近的选择性标记基因来监测哺乳动物细胞中端粒丢失的后果的测定法。通过在I-SceI内切核酸酶表达后在邻近端粒的I-SceI位点处引入双链断裂来诱导端粒损失。大多数丢失端粒的小鼠胚胎干细胞在I-Scel位点上直接添加了端粒;然而，在不将端粒添加到断裂染色体末端的细胞中观察到涉及B/F/B循环的姐妹染色单体融合和染色体不稳定性。本提案将使用该系统来解决哺乳动物细胞中端粒丢失的后果。具体目标1将研究端粒酶和Pif 1解旋酶在染色体愈合中的作用。酵母中的Pif 1突变体具有600倍更高的染色体愈合发生率，因此，Pif 1已被提议负调节染色体愈合以防止末端缺失。这些实验将检验端粒酶和Pifl在哺乳动物细胞中的染色体愈合中是重要的以及染色体愈合阻止B/F/B循环的假设。具体目标2将利用含有DNA-PKcs或NBS 1突变的细胞系，以解决非同源末端连接和Mre 11/Rad 50/Nbs 1复合物在端粒维持、染色体愈合和染色体融合中的作用。具体目标3将通过在体内表达I-Scel并监测由含有端粒I-Scel位点的15号染色体上的c-Myc基因的扩增/重排引起的癌前变化来检验由于端粒丢失引起的染色体不稳定性促进致癌的假设。

DESCRIPTION (provided by applicant): A major function of telomeres is to protect the ends of chromosomes and prevent chromosome fusion. Chromosome fusion can result in chromosome instability through breakage/fusion/bridge (B/F/B) cycles, which occur when chromosomes repeatedly break and fuse with each cell division. B/F/B cycles can be prevented or terminated by the addition of telomeres to the ends of broken chromosomes, which results in the formation of terminal deletions. In Tetrahymena and yeast, telomeres are added on to the ends of broken chromosomes by telomerase, termed chromosome healing. Chromosome rearrangements associated with telomere loss and B/F/B cycles have been found in a variety of human genetic diseases, and are thought to play an important role in the chromosome instability associated with cancer. We have established an assay that utilizes selectable marker genes located adjacent to a telomere to monitor the consequences of telomere loss in mammalian cells. Telomere loss is induced through the introduction of a double-strand break at an I-Scel site adjacent to the telomere following the expression of the I-Scel endonuclease. Most mouse embryonic stem cells that lose a telomere have telomeres added directly on at the I-Scel site; however, sister chromatid fusion and chromosome instability involving B/F/B cycles is observed in cells that do not add a telomere on to the end of the broken chromosome. The present proposal will use this system to address the consequences of telomere loss in mammalian cells. Specific Aim 1 will investigate the role of telomerase and the Pif1 helicase in chromosome healing. Pif1 mutants in yeast have a 600-fold higher incidence of chromosome healing, and as a result, Pif1 has been proposed to negatively regulate chromosome healing to prevent terminal deletions. These experiments will test the hypotheses that telomerase and Pifl are important in chromosome healing in mammalian cells and that chromosome healing prevents B/F/B cycles. Specific Aim 2 will utilize cell lines containing mutations in DNA-PKcs or NBS1, to address the role of nonhomologous end joining and the Mre11/Rad50/Nbs1 complex in telomere maintenance, chromosome healing and chromosome fusion. Specific Aim 3 will test the hypothesis that chromosome instability due to telomere loss promotes carcinogenesis by expressing I-Scel in vivo and monitoring preneoplastic changes resulting from the amplification/rearrangement of the c-Myc gene on the chromosome 15 containing a telomeric I-Scel site.