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?
Chromosomal translocations can result in many types of genetic developmental disorders as well as malignancies, yet are poorly understood. This project lays the foundation for not only predicting translocation occurrence but also for preventing them from occurring in the first place.
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