Mechanisms of formation of terminal deletions

描述(申请人提供)：端粒的一个主要功能是保护染色体末端，防止染色体融合。染色体融合可以通过断裂/融合/桥(B/F/B)循环导致染色体不稳定，B/F/B周期发生在染色体反复断裂并与每个细胞分裂融合时。可以通过在断裂的染色体末端添加端粒来防止或终止B/F/B周期，这会导致末端缺失的形成。在四膜虫和酵母菌中，端粒通过端粒酶添加到断裂的染色体末端，称为染色体愈合。与端粒丢失和B/F/B循环相关的染色体重排在多种人类遗传病中被发现，并被认为在与癌症相关的染色体不稳定性中起着重要作用。我们已经建立了一种利用位于端粒附近的可选择标记基因来监测哺乳动物细胞端粒丢失的后果的方法。端粒丢失是通过在I-SCEL内切酶表达后，在与端粒相邻的I-SCEL位点引入双链断裂而引起的。大多数失去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，在没有在断裂的染色体末端添加端粒的细胞中，观察到姐妹染色单体融合和涉及B/F/B周期的染色体不稳定。目前的建议将使用这一系统来解决哺乳动物细胞端粒丢失的后果。具体目标1将研究端粒酶和Pif1解旋酶在染色体愈合中的作用。酵母中的Pif1突变体具有高600倍的染色体愈合率，因此，Pif1已被提出负向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在哺乳动物细胞染色体修复中的重要作用，以及染色体修复阻止B/F/B周期的假设。特殊目的2将利用含有DNA-PKcs或NBS1突变的细胞系，研究非同源末端连接和Mre11/Rad50/Nbs1复合体在端粒维持、染色体修复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并监测含有端粒I-SCEL位点的15号染色体上c-Myc基因的扩增/重排引起的癌前变化，来验证端粒丢失导致的染色体不稳定性促进癌症发生的假设。

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.