POLQ- and CtIP-regulated telomere fusions and translocations are involved in early events in carcinogenesis

项目总结 我们建议研究调节细胞逃脱危机能力的机制 端粒缩短。随着正常人类细胞的衰老，它们的端粒逐渐变短。当端粒 显著缩短的是，细胞经历衰老，这是一种自然产生的抗癌屏障。如果， 然而，细胞应该遭受转化突变，它可以绕过衰老，继续增殖。 直到它的端粒变得太短而不起作用。由此导致的终端保护不足引发了 “危机”，指染色体断裂时基因组不稳定的状态：融合：桥接 几乎总是导致细胞死亡的循环。在极少数情况下，细胞可以分解其融合， 重建其端粒并稳定其基因组。这种细胞据说是永生的，很可能是 它们是大多数人类癌症的始祖。端粒维持的(Dys)调节也是 在其他实验系统中与衰老、永生化和肿瘤形成相关的因素增加了信心 相信这些问题是保守的和重要的。此前，我们已经证明了DNA连接酶III 和聚(ADP)核糖聚合酶1是人类细胞在危机中生存所必需的。在这里，我们建议定义 DNA聚合酶theta/q(POLQ)在这一过程中的作用。出乎意料的是， 我们发现，POLQ的缺失会导致端粒延长并逃脱危机。我们将揭开如何 POLQ通常会抑制这些事件。 生存危机的一个组成部分是解决发生的染色体融合/易位的要求 在危机期间。这样做的一种方式是通过施加张力(又名， “破损”)。然而，这一过程具有高度的诱变性，往往会导致致命的后果。一秒钟 更有可能确保生存的解决过程是将染色体融合转化为超精细的桥梁 (不明飞行物)，然后酶法-在一个非常鲜为人知的过程-分解这些不明飞行物。在这里，我们 证明切除核酸酶C端相互作用蛋白(CtIP)的功能丧失会导致 高频率的未解决的不明飞行物，我们建议进行实验，以机械地解开 这些桥，其中许多涉及端粒，是产生的，以及为什么它们没有被解析。在所有这些中 我们利用亨德里克森和贝尔德实验室的优势。亨德里克森实验室 擅长基因打靶技术，研究基因功能丧失突变(POLQ)的影响 和在这种情况下的CtIP)。贝尔德实验室在分析方面处于世界领先地位 经历危机的人类细胞中的端粒融合事件。他们能够刻画单项运动的动态 端粒末端使该领域对人类端粒融合的机制有了最深入的了解 细胞。综上所述，我们提出的研究对DNA修复和端粒维持的影响及其重要性 对癌症生物学的这些过程的理解是显而易见的。

PROJECT SUMMARY We propose to investigate the mechanisms that regulate a cell’s ability to escape from the crisis caused by telomere shortening. As normal human cells age, their telomeres gradually shorten. When the telomeres shorten significantly, the cell undergoes senescence, which is a naturally occurring barrier to cancer. If, however, a cell should suffer a transforming mutation, it can by-pass senescence and continue to proliferate until its telomeres become so short that they are non-functional. The resulting lack of end protection triggers “crisis”, a state that is highlighted by genomic instability as chromosomes engage in breakage:fusion:bridging cycles that almost invariably result in the death of the cell. On rare occasions a cell can resolve its fusions, reestablish its telomeres and stabilize its genome. Such cells are said to be immortalized and it is likely that they are the progenitors of most human cancers. That the (dys)regulation of telomere maintenance is also associated with aging, immortalization, and tumorigenesis in other experimental systems adds confidence to the belief that these issues are conserved and important. Previously, we have demonstrated that DNA ligase III and poly (ADP) ribose polymerase 1 are required for human cells to survive crisis. Here, we propose to define the role of DNA polymerase theta/Q (POLQ), which acts in the same pathway, in this process. Unexpectedly, we show that deletion of POLQ causes telomere elongation and escape from crisis. We will uncover how POLQ normally suppresses these events. Integral to surviving crisis is a requirement to resolve the chromosomal fusions/translocations that occurred during crisis. One way to do this is to physically shear them apart by the application of tension (aka, “breakage”). This process, however, is highly mutagenic and often leads to lethal outcomes. A second resolution process more likely to ensure survival is to convert the chromosome fusions into ultra-fine bridges (UFBs) and then enzymatically — in a process that is very poorly understood — resolve these UFBs. Here, we demonstrate that the loss-of-function of the resection nuclease, C-terminal interacting protein (CtIP), results in a high frequency of UFBs that are not resolved and we propose experiments to mechanistically unravel how these bridges, many of which involve telomeres, are generated and why they are not resolved. In all of these approaches we utilize the strengths of the Hendrickson and Baird laboratories. The Hendrickson laboratory excels at the technology of gene targeting to study the impact of loss-of-function mutations of genes (POLQ and CtIP in this instance) on telomere maintenance. The Baird laboratory is the world’s leader in analyzing telomere fusion events in human cells undergoing crisis. Their ability to characterize the dynamics of single telomeric ends has provided the field’s deepest understanding of the mechanism of telomere fusions in human cells. In summary, our proposed studies impact on DNA repair and telomere maintenance and the importance of understanding these processes for cancer biology is clear.