Mechanisms for Chromosomal Translocations

 描述(申请人提供)：染色体易位，即一条染色体的一段与一条异源染色体相连，可导致胎儿发育异常或多种恶性肿瘤。要发生染色体易位，必须有：1)在异源染色体上同时发生双链断裂(DSB)，以及2)DSB重新连接到异源而不是同源的染色体自由端。如果细胞在易位中存活下来，它在胎儿发育过程中出现异常分化或肿瘤转化的风险很大。尽管它在DNA动力学和疾病中很重要，但染色体易位的机制尚不清楚。DNA双链断裂可通过三种途径修复：同源重组(HR)、单链退火法(SSA)和非同源末端连接(NHEJ)。一些证据，如癌症易位连接的测序，表明易位主要是通过NHEJ形成的。有两条主要的NHEJ途径，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组分，如Metnase、Ku80和Ligase 4，抑制了易位。另一方面，最近我们和其他人发现，PARP1、CtIP和DNA连接酶3等aNHEJ组分促进了染色体易位。当PARP1成功地与Ku复合体竞争DSB的游离DNA末端时，ANHEJ被启动。我们发现，使用临床相关的抑制剂olaparib和rucaparib或siRNA抑制PARP1可以防止多种易位报告系统中的染色体易位。此外，PARP1抑制抑制了电离辐射或VP16在正常人类成纤维细胞和小鼠造血细胞中产生的易位。尽管它在易位中很重要，但aNHEJ的机制和成分仍然不清楚。我们用免疫沉淀(IP)和质谱仪鉴定了PARP1下游的一个NHEJ中的两个新的成分：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，对aNHEJ和易位是必不可少的。2)进一步，我们发现了一种新的5‘端核酸酶EEPD1，它对HR和aNHEJ都是必需的，可能是因为它促进了5’端切除。羟基脲发现它与PARP1相关后，EEPD1相互作用的质谱图。定义这些新的PARP1下游伙伴为aNHEJ的机制提供了新的线索，从而揭示了染色体易位的机制。本应用将从三个方面剖析PARP1如何通过Pso4和EEPD1启动aNHEJ级联反应：目的1)PARP1促进aNHEJ和易位的机制是什么？目的2)PARP1合作伙伴Pso4是如何介导NHEJ和易位的？目的3)PARP1相关的5‘核酸酶EEPD1是如何介导NHEJ和易位的？

 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?