Ligase III regulates survival from crisis induced by gradual telomere shortening

连接酶 III 调节端粒逐渐缩短引起的危机中的生存

• 批准号: 9308903
• 负责人: ERIC A HENDRICKSON
• 金额: $ 33.52万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9308903
• 关键词: Address; Affect; Aging; Alpha Cell; Applications Grants; Area; Award; Belief; Binding; Biological Models; Biology; Cancer Biology; Cell Death; Cell Line; Cell physiology; Cells; Chromatin; Chromosomal translocation; Chromosomes; DNA; DNA Double Strand Break; DNA Repair; DNA ligase III; Data; Deposition; Disease; Double Strand Break Repair; Dyskeratosis Congenita; Event; Gene Amplification; Gene Mutation; Gene Targeting; Genes; Genetic; Genome; Genomic Instability; Histones; Human; Human Cell Line; Investigation; Knock-out; Knowledge; Laboratories; Lead; Ligase; Link; Maintenance; Malignant Neoplasms; Mediating; Medicine; Mental Retardation; Modeling; Mus; Mutation; Nobel Prize; Nonhomologous DNA End Joining; Null Lymphocytes; Pancytopenia; Pathologic; Pathway interactions; Physiology; Play; Poly(ADP-ribose) Polymerases; Prediabetes syndrome; Predisposition; Process; Production; Proliferating; Regulation; Role; Sister Chromatid; Somatic Cell; Structure; Syndrome; System; TP53 gene; Technology; Telomerase; Telomere Maintenance; Telomere Shortening; Testing; Variant; XRCC1 gene; alpha-Thalassemia; base; cell age; cell transformation; chromatin remodeling; conditional mutant; genome-wide; genome-wide analysis; homologous recombination; insight; loss of function; loss of function mutation; nondeletion type alpha-thalassemia/mental retardation syndrome; prevent; progenitor; public health relevance; repaired; response; senescence; single molecule; telomere; tumorigenesis

 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.

 描述(申请人提供)：我们建议研究调节细胞从端粒逐渐缩短引起的危机中逃脱的机制(S)。特别是，我们将确定DNA DSB(双链断裂)修复的A-NHEJ(替代-非同源末端连接)途径以及II)染色质重塑基因ATRX(X连锁的阿尔法地中海贫血/智力低下综合征)在这一过程中所起的作用。随着正常人类细胞的衰老，它们的端粒逐渐变短。当端粒显著缩短时，细胞经历衰老，这是一种自然产生的、对癌症的非增殖性屏障。然而，如果一个细胞遭受转化突变，它可以绕过衰老，继续增殖，直到它的端粒变得如此之短，以至于它们不起作用。由此导致的末端保护的缺乏触发了“危机”，这种状态突出表现为基因组的不稳定，因为染色体参与了断裂：融合：桥接循环，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。这种细胞据说是永生化的，很可能是 大多数人类癌症的始祖。在其他实验系统中，端粒维持(Dys)的调节也与衰老、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LIGIII(DNA连接酶III)和PARP1(聚(ADP)核糖聚合酶1)基因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PARP1在DNA DSB修复A-NHEJ分支中的作用我们假设是A-NHEJ的缺乏导致了经历危机的细胞的死亡，我们建议：一)使用结构：功能方法来定义这一过程所需的分子相互作用；二)使用定向方法和全基因组筛选来确定参与危机生存的其他基因；三)开始测试LIGIII如何机械地控制这一过程的模型。在……里面 此外，我们描述了我们的初步数据，证明ATRX是ALT(端粒替代延长)的关键调节因子，并描述了一个实验系统，在该系统中我们可以研究ALT的发生。在所有这些方法中，我们利用了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于基因打靶技术，研究基因功能丧失突变(在这种情况下是LIGIII、PARP1和ATRX)对端粒维持的影响。基因打靶的使用提供了一种简便的实验系统，在该系统中可以快速地将空的、亚形态的和/或条件突变引入人类体细胞。贝尔德实验室在分析处于危机中的人类细胞的端粒融合事件方面处于世界领先地位。他们能够描述单个端粒末端的动力学特征，这为该领域提供了对人类细胞端粒融合机制的最深入了解。综上所述，我们提出的研究对DNA修复和端粒维持有影响，了解这些过程对癌症生物学的重要性是显而易见的。