Mechanisms for Chromosomal Translocations

染色体易位的机制

• 批准号: 9029327
• 负责人: Robert A Hromas
• 金额: $ 28.88万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-13 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9029327
• 关键词: Apoptosis; Cells; Chemotherapy-Oncologic Procedure; Chromosomal translocation; Chromosomes; Chromosomes, Human, Pair 2; Clinical; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Ligases; DNA replication fork; Data; Development; Disease; Double Strand Break Repair; Excision; Exonuclease; Fetal Development; Fibroblasts; Foundations; G22P1 gene; Genetic; Health; Hematopoietic; Hereditary Disease; Histones; Human; Human Genetics; Immunoprecipitation; Inherited; Ionizing radiation; Ligase; Ligation; Light; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mitotic; Molecular; Mono-S; Mus; Neoplastic Cell Transformation; Nonhomologous DNA End Joining; Oncogenic; Pathway interactions; Phosphoric Monoester Hydrolases; Play; Proteins; Recruitment Activity; Reporter; Repression; Resected; Risk; Role; Sister Chromatid; Small Interfering RNA; System; VP 16; XRCC5 gene; chromosomal location; clinically relevant; developmental disease; endonuclease; fetal; high risk; homologous recombination; hydroxyurea; improved; in vivo; inhibitor/antagonist; novel; nuclease; prevent; repaired; seal; ubiquitin-protein ligase

 DESCRIPTION (provided by applicant): Chromosomal translocations, where a segment from one chromosome is joined to a heterologous chromosome, can result in fetal developmental abnormalities or a myriad of malignancies. For a chromosomal translocation to occur there must be: 1) simultaneous double strand breaks (DSBs) on heterologous chromosomes, and 2) re-ligation of the DSBs to heterologous and not homologous chromosomal free ends. Should the cell survive a translocation, it is at great risk for abnormal differentiation during fetal development, or for neoplastic transformation. Despite its importance in DNA dynamics and disease, the mechanisms of chromosomal translocations are not clear. DNA DSBs can be repaired by three pathways: homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). Several lines of evidence, such as sequencing cancer translocation junctions, indicate that translocations were predominantly formed via NHEJ. There are two major NHEJ pathways, the more common classical (cNHEJ) pathway, and the alternative (aNHEJ) pathway. Surprisingly, we and others discovered that cNHEJ components, such as Metnase, Ku80, and Ligase 4, suppressed translocations. On the other hand, recently we and others found that aNHEJ components such as PARP1, CtIP, and DNA Ligase 3 promote chromosomal translocations. ANHEJ is initiated when PARP1 successfully competes with the Ku complex for the free DNA ends of a DSB. We found that PARP1 repression with the clinically relevant inhibitors olaparib and rucaparib, or siRNA, could prevent chromosomal translocations in multiple translocation reporter systems. In addition, PARP1 inhibition repressed ionizing radiation- or VP16-generated translocations in normal human fibroblast and murine hematopoietic cells. Despite its importance in translocations, the mechanism and components of aNHEJ remain undefined. We have identified two novel components in aNHEJ downstream of PARP1 using immunoprecipitation (IP) and mass spectroscopy: 1) We have discovered that the E3 ubiquitin ligase, Pso4 (also termed Prp19) associates with PARP1 after ionizing radiation, and is essential for aNHEJ and translocations. 2) Further, we identified a novel 5' nuclease, EEPD1 that is also essential for both HR and aNHEJ, likely by its enhancement of 5' end resection. Mass spectroscopy of EEPD1 interactions after hydroxyurea found it associated with PARP1. Defining these novel PARP1 downstream partners has shed new light into the mechanisms of aNHEJ and therefore chromosomal translocations. This application will dissect how PARP1 initiates the cascade of aNHEJ through Pso4 and EEPD1 in three aims: Aim 1) What are the mechanisms by which PARP1 promotes aNHEJ and translocations? Aim 2) How does the PARP1 partner Pso4 mediate aNHEJ and translocations? Aim 3) How does the PARP1-associated 5' nuclease EEPD1 mediate aNHEJ and translocations?

 描述（由应用程序提供）：染色体翻译，其中一个染色体的片段与异源染色体相结合，可能导致胎儿发育异常或无数的恶性肿瘤。为了使染色体易位发生：1）在异源染色体上同时双链断裂（DSB），以及2）将DSB的重新连接到异源，而不是同源染色体自由端。如果细胞存活一个易位，则在胎儿发育期间或肿瘤转化时有异常分化的风险。尽管它在DNA动力学和疾病中的重要性，但染色体易位的机制尚不清楚。 DNA DSB可以通过三种途径来修复：同源重组（HR），单链退火（SSA）和非理论末端连接（NHEJ）。几条证据，例如测序癌症易位连接，表明易位主要是通过NHEJ形成的。有两个主要的NHEJ途径，更常用的经典（CNHEJ）途径，还有替代（ANHEJ）途径。令人惊讶的是，我们和其他人发现CNHEJ组件（例​​如metnase，ku80和连接酶4）抑制了易位。另一方面，最近我们和其他人发现，诸如PARP1，CTIP和DNA连接酶3之类的Anhej成分促进了染色体翻译。当PARP1成功与KU综合体竞争DSB的自由DNA末端时，就开始了Anhej。我们发现，具有临床相关抑制剂Olaparib和Rucaparib或siRNA的PARP1表达可以防止多个转移报告基因系统中的染色体翻译。此外，PARP1抑制反映了正常人成纤维细胞和鼠造血细胞中的电离辐射或VP16产生的易位。尽管它在易位方面具有重要意义，但Anhej的机制和组成部分仍然不确定。我们已经使用免疫沉淀（IP）和质谱法确定了PARP1下游的两个新成分：1）我们发现，E3泛素连接酶PSO4（也称为PRP19）与PARP1的同伴在电离辐射后与PARP1相关，并且对于ANHEJ和翻译是必不可少的。 2）此外，我们确定了一种新颖的5'核酸酶，EEPD1，这对于HR和Anhej也是必不可少的，这可能是由于它增强了5'末端切除术。羟基脲后EEPD1相互作用的质谱发现与PARP1相关。定义这些新颖的PARP1下游伴侣已为Anhej的机制和染色体易位的机理提供了新的光。该应用程序将在三个目标中剖析PARP1如何通过PSO4和EEPD1启动Anhej的级联反应：AIM 1）PARP1促进Anhej和转运的机制是什么？目标2）PARP1合作伙伴PSO4媒体ANHEJ和易位如何？目标3）PARP1相关的5'核酸酶EEPD1如何介导Anhej和易位？