Mechanisms for Chromosomal Translocations

 描述（由申请人提供）：染色体易位，其中来自一条染色体的片段连接到异源染色体，可导致胎儿发育异常或多种恶性肿瘤。为了发生染色体易位，必须存在：1）异源染色体上的同时双链断裂（DSB），和2）DSB与异源而非同源染色体游离末端的再连接。如果细胞在易位中存活，则在胎儿发育期间异常分化或肿瘤转化的风险很大。尽管它在DNA动力学和疾病中的重要性，染色体易位的机制尚不清楚。DNA双链断裂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，如测序癌症易位连接，表明易位主要通过NHEJ形成。存在两种主要的NHEJ途径，更常见的经典（cNHEJ）途径和替代（aNHEJ）途径。令人惊讶的是，我们和其他人发现cNHEJ组分，如Metnase，Ku 80和Ligase 4，抑制易位。另一方面，最近我们和其他人发现aNHEJ组分如PARP 1、CtIP和DNA连接酶3促进染色体易位。当PARP 1成功地与Ku复合物竞争DSB的游离DNA末端时，ANHEJ被启动。我们发现用临床相关抑制剂olaparib和rucaparib或siRNA抑制PARP 1可以防止多易位报告系统中的染色体易位。此外，PARP 1抑制抑制电离辐射-或VP 16-正常人成纤维细胞和小鼠造血细胞中产生的易位。尽管其在易位中的重要性，但aNHEJ的机制和组分仍不明确。我们已经使用免疫沉淀（IP）和质谱鉴定了PARP 1下游aNHEJ中的两种新组分：1）我们已经发现E3泛素连接酶Pso 4（也称为Prp 19）在电离辐射后与PARP 1缔合，并且对于aNHEJ和易位是必需的。2)此外，我们鉴定了一种新的5'核酸酶EEPD 1，其也是HR和aNHEJ所必需的，可能是通过其5'端切除的增强。羟基脲后EEPD 1相互作用的质谱发现它与PARP 1相关。定义这些新的PARP 1下游伙伴为aNHEJ的机制以及染色体易位提供了新的线索。本申请将剖析PARP 1如何通过Pso 4和EEPD 1启动aNHEJ的级联反应，目的有三：目的1）PARP 1促进aNHEJ和易位的机制是什么？目的2）PARP 1伴侣Pso 4如何介导aNHEJ和易位？目的3）PARP 1相关的5'核酸酶EEPD 1如何介导aNHEJ和易位？

 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?