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

项目摘要 我们建议研究调节细胞逃避危机能力的机制， 端粒缩短随着正常人类细胞的衰老，它们的端粒逐渐缩短。当端粒 当细胞显著缩短时，细胞经历衰老，这是癌症的天然屏障。如果， 然而，细胞应该经历转化突变，它可以绕过衰老并继续增殖， 直到它的端粒变短而失去功能。由此产生的缺乏末端保护触发 “危机”，一种状态，是突出的基因组不稳定性，因为染色体从事断裂：融合：桥接 几乎总是导致细胞死亡的周期。在极少数情况下，细胞可以解决它的融合， 重建端粒并稳定基因组。这种细胞被认为是永生化的， 它们是大多数人类癌症的祖先。端粒维持的（dys）调节也是 在其他实验系统中与衰老，永生化和肿瘤发生相关的研究增加了对 相信这些问题是保守的和重要的。以前，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，它在同一途径中起作用。没想到， 我们发现POLQ的缺失导致端粒延长并使端粒脱离危象。我们将揭开 POLQ通常会抑制这些事件。 生存危机的必要条件是解决发生的染色体融合/易位 在危机中。一种方法是通过施加张力将它们物理剪切开（又名， “断裂”）。然而，这个过程是高度致突变的，并且常常导致致命的结果。第二 一个更有可能确保存活的解决方法是将染色体融合转化为超细桥 （UFB），然后酶-在一个非常不了解的过程中-解决这些UFB。这里我们 证明切除核酸酶C-末端相互作用蛋白（CtIP）的功能丧失导致 高频率的UFB没有得到解决，我们提出实验来机械地解开如何 这些桥梁，其中许多涉及端粒，产生和为什么他们没有解决。在所有这些 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，研究基因功能缺失突变（POLQ）的影响 和CtIP）对端粒维持的影响。贝尔德实验室是世界上分析 端粒融合事件在人类细胞经历危机。他们的能力，以表征动态的单一 端粒末端的研究提供了该领域对人类端粒融合机制的最深刻理解。 细胞总之，我们提出的研究对DNA修复和端粒维持的影响以及 了解这些癌症生物学过程的方法是显而易见的。

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.