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?

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM109645-02
Application #
9029327
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Willis, Kristine Amalee
Project Start
2015-03-13
Project End
2018-11-30
Budget Start
2015-12-01
Budget End
2016-11-30
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Florida
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Shao, Lijian; Chang, Jianhui; Feng, Wei et al. (2018) The Wave2 scaffold Hem-1 is required for transition of fetal liver hematopoiesis to bone marrow. Nat Commun 9:2377
Wang, Weibin; Daley, James M; Kwon, Youngho et al. (2018) A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1-Dna2 in long-range DNA end resection. J Biol Chem 293:17061-17069
Narayan, Satya; Jaiswal, Aruna S; Sharma, Ritika et al. (2017) NSC30049 inhibits Chk1 pathway in 5-FU-resistant CRC bulk and stem cell populations. Oncotarget 8:57246-57264
Kim, Hyun-Suk; Nickoloff, Jac A; Wu, Yuehan et al. (2017) Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks. J Biol Chem 292:2795-2804
Srinivasan, Gayathri; Sidhu, Gurjit Singh; Williamson, Elizabeth A et al. (2017) Synthetic lethality in malignant pleural mesothelioma with PARP1 inhibition. Cancer Chemother Pharmacol 80:861-867
Nickoloff, Jac A; Jones, Dennie; Lee, Suk-Hee et al. (2017) Drugging the Cancers Addicted to DNA Repair. J Natl Cancer Inst 109:
Kim, Hyun-Suk; Williamson, Elizabeth A; Nickoloff, Jac A et al. (2017) Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks. J Biol Chem 292:1414-1425
Chun, Changzoon; Wu, Yuehan; Lee, Suk-Hee et al. (2016) The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis. Cell Cycle 15:957-62
Narayan, Satya; Jaiswal, Aruna S; Law, Brian K et al. (2016) Interaction between APC and Fen1 during breast carcinogenesis. DNA Repair (Amst) 41:54-62
Sullivan, Katherine; Cramer-Morales, Kimberly; McElroy, Daniel L et al. (2016) Identification of a Small Molecule Inhibitor of RAD52 by Structure-Based Selection. PLoS One 11:e0147230

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