Mechanisms for Chromosomal Translocations

染色体易位的机制

• 批准号: 9187481
• 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/9187481
• 关键词: Apoptosis; Cells; Chemotherapy-Oncologic Procedure; Chromosomal translocation; Chromosome abnormality; Chromosomes; Chromosomes, Human, Pair 2; Clinical; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Ligases; DNA replication fork; Data; Development; Disease; Excision; Exonuclease; Fetal Development; Fibroblasts; Foundations; G22P1 gene; Hematopoietic; Hereditary Disease; Histones; Human; Human Genetics; Immunoprecipitation; Inherited; Intuition; 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; Proteins; Recruitment Activity; Reporter; Repression; Risk; Role; Sister Chromatid; Small Interfering RNA; System; VP 16; XRCC5 gene; chromosomal location; clinically relevant; developmental disease; developmental genetics; endonuclease; fetal; high risk; homologous recombination; hydroxyurea; improved; in vivo; inhibitor/antagonist; novel; nuclease; prevent; public health relevance; 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（由申请人提供）：染色体易位，即一条染色体的一个片段与异源染色体连接，可能导致胎儿发育异常或多种恶性肿瘤。要发生染色体易位，必须：1) 异源染色体上同时发生双链断裂 (DSB)，2) DSB 与异源而非同源染色体游离端重新连接。如果细胞在易位后存活下来，那么它在胎儿发育过程中就面临着异常分化或肿瘤转化的巨大风险。尽管染色体易位在 DNA 动力学和疾病中很重要，但其机制尚不清楚。 DNA DSB 可以通过三种途径修复：同源重组 (HR)、单链退火 (SSA) 和非同源末端连接 (NHEJ)。一些证据，例如癌症易位连接点的测序，表明易位主要是通过 NHEJ 形成的。 NHEJ 途径主要有两种，即更常见的经典途径 (cNHEJ) 和替代途径 (aNHEJ)。令人惊讶的是，我们和其他人发现 cNHEJ 成分，例如 Metnase、Ku80 和 Ligase 4，可以抑制易位。另一方面，最近我们和其他人发现 PARP1、CtIP 和 DNA 连接酶 3 等 aNHEJ 成分可促进染色体易位。当 PARP1 成功与 Ku 复合物竞争 DSB 的游离 DNA 末端时，ANHEJ 就会启动。我们发现，用临床相关抑制剂olaparib和rucaparib（或siRNA）抑制PARP1可以防止多个易位报告系统中的染色体易位。此外，PARP1 抑制可抑制正常人成纤维细胞和小鼠造血细胞中电离辐射或 VP16 产生的易位。尽管 aNHEJ 在易位中很重要，但其机制和组成部分仍不清楚。我们使用免疫沉淀 (IP) 和质谱法鉴定了 PARP1 下游 aNHEJ 中的两个新成分：1) 我们发现 E3 泛素连接酶 Pso4（也称为 Prp19）在电离辐射后与 PARP1 结合，并且对于 aNHEJ 和易位至关重要。 2) 此外，我们还发现了一种新的 5' 核酸酶 EEPD1，它对于 HR 和 aNHEJ 也至关重要，可能是通过它增强了 5' 末端切除。羟基脲后 EEPD1 相互作用的质谱发现它与 PARP1 相关。定义这些新的 PARP1 下游伙伴为 aNHEJ 以及染色体易位的机制提供了新的线索。本申请将剖析 PARP1 如何通过 Pso4 和 EEPD1 启动 aNHEJ 级联，实现三个目标： 目标 1) PARP1 促进 aNHEJ 和易位的机制是什么？目标 2) PARP1 伙伴 Pso4 如何介导 aNHEJ 和易位？目标 3) PARP1 相关 5' 核酸酶 EEPD1 如何介导 aNHEJ 和易位？