Ligase III regulates survival from crisis induced by gradual telomere shortening

 描述（由申请人提供）：我们建议研究调节细胞逃避由逐渐端粒缩短引起的危机的能力的机制。特别是，我们将定义的作用，i）A-NHEJ（交替非同源末端连接）DNA DSB（双链断裂）修复途径，以及ii）染色质重塑基因，ATRX（α地中海贫血/精神发育迟滞综合征，X连锁），在这一过程中发挥作用。随着正常人类细胞的衰老，它们的端粒逐渐缩短。当端粒显著缩短时，细胞经历衰老，这是一种自然发生的非增殖性癌症屏障。然而，如果一个细胞发生了转化突变，它可以绕过衰老，继续增殖，直到它的端粒变得如此之短，以至于它们失去功能。由此导致的末端保护的缺乏引发了“危机”，这是一种由基因组不稳定性所突出的状态，因为染色体参与断裂：融合：桥接周期，几乎总是导致细胞死亡。在极少数情况下，细胞可以重建其端粒并稳定其基因组。这种细胞被认为是永生化的，它们很可能是 大多数人类癌症的祖先。在其他实验系统中，端粒维持的（dys）调节也与衰老、永生化和肿瘤发生有关，这增加了人们对这些问题是保守和重要的信念的信心。在这里，我们证明了基因LIGIII（DNA连接酶III）和PARP 1 {聚（ADP）核糖聚合酶1}是人类细胞在端粒逐渐缩短引起的危机中生存所必需的。LIGIII和PARP 1在DNA DSB修复的A-NHEJ分支中起作用。我们假设是A-NHEJ的缺乏导致经历危象的细胞死亡，并且我们提出i）使用结构：功能方法来定义该过程所需的分子相互作用，ii）使用定向方法和全基因组筛选来鉴定参与危象存活的其他基因，以及iii）开始测试LIGIII如何机械地控制该过程的模型。在 此外，我们描述了我们的初步数据表明，ATRX是一个重要的调节ALT（选择性延长端粒），我们描述了一个实验系统，我们可以研究ALT的起源。在所有这些方法中，我们利用了Hendrickson和Baird实验室的优势。Hendrickson实验室擅长于基因靶向技术，以研究基因（在这种情况下，LIGIII，PARP 1和ATRX）功能丧失突变对端粒维持的影响。基因靶向的使用提供了一种简单的实验系统，其中可以将无效突变、亚型突变和/或条件突变快速引入人体细胞。贝尔德实验室是分析人类细胞中端粒融合事件的世界领导者。他们能够表征单个端粒末端的动力学，这为该领域对人类细胞中端粒融合机制的理解提供了最深刻的理解。总之，我们提出的研究对DNA修复和端粒维持的影响，以及理解这些过程对癌症生物学的重要性是显而易见的。

 DESCRIPTION (provided by applicant): We propose to investigate the mechanism(s) that regulate a cell's ability to escape from the crisis caused by gradual telomere shortening. In particular, we will define the roles that the i) A-NHEJ (alternative-non- homologous end joining) pathway of DNA DSB (double-strand break) repair as well as the ii) chromatin remodeling gene, ATRX (alpha thalassemia/mental retardation syndrome, X-linked), play in this process. As normal human cells age, their telomeres gradually shorten. When the telomeres shorten significantly, the cell undergoes senescence, which is a naturally-occurring, non-proliferative 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 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. Here, we demonstrate that the genes LIGIII (DNA ligase III) and PARP1 {poly(ADP) ribose polymerase 1} are required for human cells to survive the crisis induced by gradual telomere shortening. LIGIII and PARP1 function in the A-NHEJ branch of DNA DSB repair. We hypothesize that it is the absence of A- NHEJ that results in the death of cells undergoing crisis and we propose to i) use structure:function approaches to define the molecular interactions required for the process, ii) identify other genes involved in crisis survival using directed approaches and genome-wide screens and iii) begin to test models for how LIGIII might mechanistically control this process. In addition, we describe our preliminary data demonstrating that ATRX is a crucial regulator of ALT (alternative lengthening of telomeres) and we describe an experimental system in which we can study the genesis of ALT. 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 (LIGIII, PARP1 and ATRX in this instance) on telomere maintenance. The use of gene targeting provides a facile experimental system in which null, hypomorphic, and/or conditional mutations can be introduced with rapidity into human somatic cells. 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.