Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.

• Chromosomal translocations are mediated by PARP1 and can be suppressed by the clinical PARP1 inhibitors.Chromosomal translocations are common contributors to malignancy, yet little is known about the precise molecular mechanisms by which they are generated. Sequencing translocation junctions in acute leukemias revealed that the translocations were likely mediated by a DNA double-strand break repair pathway termed nonhomologous endjoining (NHEJ). There are major 2 types of NHEJ: (1) the classical pathway initiated by the Ku complex, and (2) the alternative pathway initiated by poly ADP-ribose polymerase 1 (PARP1). Recent reports suggest that classical NHEJ repair components repress translocations, whereas alternative NHEJ components were required for translocations. The rate-limiting step for initiation of alternative NHEJ is the displacement of the Ku complex by PARP1. Therefore, we asked whether PARP1 inhibition could prevent chromosomal translocations in 3 translocation reporter systems. We found that 2 PARP1 inhibitors or repression of PARP1 protein expression strongly repressed chromosomal translocations, implying that PARP1 is essential for this process. Finally, PARP1 inhibition also reduced both ionizing radiation-generated and VP16-generated translocations in 2 cell lines. These data define PARP1 as a critical mediator of chromosomal translocations and raise the possibility that oncogenic translocations occurring after high-dose chemotherapy or radiation could be prevented by treatment with a clinically available PARP1 inhibitor. (Blood. 2013;121(21):4359-4365) IntroductionChromosomal translocations both classify types of malignancies and are required for the origin of those malignancies.1,2 Because of this trend, translocations have been widely studied, but their precise molecular mechanism remains poorly understood. It is intuitively and experimentally clear that simultaneous DNA doublestrand breaks (DSBs) must occur in distinct chromosomes for a translocation to occur.3 DNA DSBs can be repaired by 3 pathways: (1) homologous recombination (HR), where sequence integrity is preserved; or (2) single-strand annealing (SSA) and (3) nonhomologous end-joining (NHEJ), both of which generate deletions. 4 Sequencing junctions of leukemic translocations in patient samples revealed that these junctions often had deleted sequences, indicating that these translocations predominantly arose by SSA or NHEJ. 5 Therefore, HR is not thought to play a significant role in chromosomal translocations.1,2 SSA and NHEJ can be distinguished by the presence of repeated sequences adjacent to the junction site that could mediate the annealing in SSA. Thus, when long repeated sequences are adjacent to DSBs, translocations occur more frequently via SSA vs NHEJ.3 When no repeated sequences are present, then translocations are mediated by NHEJ.There are 2 major NHEJ pathways: the dominant, classical (cNHEJ) pathway; and the alternative (aNHEJ) pathway.4 cNHEJ begins when the Ku70/80 complex recognizes free DNA ends and recruits D...

Purpose The vitamin E analog γ-tocotrienol (GT3) is a powerful radioprotector. GT3 reduces post-radiation vascular peroxynitrite production, an effect dependent on inhibition of hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase. HMG-CoA reductase inhibitors mediate their pleiotropic effects via eNOS that requires the co-factor tetrahydrobiopterin (BH4). This study investigated the effects of radiation on BH4 bioavailability and of GT3 on BH4 metabolism. Methods and Materials Mice were exposed to 8.5 Gy total body irradiation (TBI). Lung BH4 and total biopterin concentrations were measured 0, 3.5, 7, 14 and 21 days after TBI using differential oxidation followed by HPLC. The effect of exogenous GT3 and BH4 treatment on post-radiation vascular oxidative stress and bone marrow colony-forming units (BM-CFU) were assessed in vivo. The effect of GT3 on endothelial cell apoptosis and endothelial expression of GTP cyclohydrolase 1 (GTPCH), GTPCH regulatory protein (GFRP), GFRP transcription, GFRP protein levels, and GFRP-CTPCH protein binding were determined in vitro. Results Compared to baseline levels, lung BH4 concentrations decreased by 24% at 3.5 days after TBI, an effect that was reversed by GT3. At 14 and 21 days after TBI, compensatory increases in BH4 (58% and 80%, respectively) were observed. Relative to vehicle-treated controls, both GT3 and BH4 supplementation reduced post-irradiation vascular peroxynitrite production at 3.5 days (by 66% and 33%, respectively), and BH4 resulted in a 68% increase in BM-CFU. GT3 ameliorated endothelial cell apoptosis and reduced endothelial GFRP protein levels and GFRP-GTPCH binding by decreasing transcription of the GFRP gene. Conclusions BH4 bioavailability is reduced in the early post-radiation phase. Exogenous administration of BH4 reduces post-irradiation vascular oxidative stress. GT3 potently reduces the expression of GFRP, one of the key regulatory proteins in the BH4 pathway, and may thus exert some of its beneficial effects on post-radiation free-radical production partly by counteracting the decrease in BH4.

Knowledge of the mechanisms involved in the radiation response is critical for developing interventions to mitigate radiation-induced injury to normal tissues. Exposure to radiation leads to increased oxidative stress, DNA-damage, genomic instability and inflammation. The transcription factor CCAAT/enhancer binding protein delta (Cebpd; C/EBPδ is implicated in regulation of these same processes, but its role in radiation response is not known. We investigated the role of C/EBPδ in radiation-induced hematopoietic and intestinal injury using a Cebpd knockout mouse model. Cebpd−/− mice showed increased lethality at 7.4 and 8.5 Gy total-body irradiation (TBI), compared to Cebpd+/+ mice. Two weeks after a 6 Gy dose of TBI, Cebpd−/− mice showed decreased recovery of white blood cells, neutrophils, platelets, myeloid cells and bone marrow mononuclear cells, decreased colony-forming ability of bone marrow progenitor cells, and increased apoptosis of hematopoietic progenitor and stem cells compared to Cebpd+/+ controls. Cebpd−/− mice exhibited a significant dose-dependent decrease in intestinal crypt survival and in plasma citrulline levels compared to Cebpd+/+ mice after exposure to radiation. This was accompanied by significantly decreased expression of γ-H2AX in Cebpd−/− intestinal crypts and villi at 1 h post-TBI, increased mitotic index at 24 h post-TBI, and increase in apoptosis in intestinal crypts and stromal cells of Cebpd−/− compared to Cebpd+/+ mice at 4 h post-irradiation. This study uncovers a novel biological function for C/EBPδ in promoting the response to radiation-induced DNA-damage and in protecting hematopoietic and intestinal tissues from radiation-induced injury.

Aims: The free radical scavenger and nitric oxide synthase cofactor, 5,6,7,, plays a well-documented role in many disorders associated with oxidative stress, including normal tissue radiation responses. Radiation exposure is associated with decreased BH4 levels, while BH4 supplementation attenuates aspects of radiation toxicity. The endogenous synthesis of BH4 is catalyzed by the enzyme guanosine triphosphate cyclohydrolase I (GTPCH1), which is regulated by the inhibitory GTP cyclohydrolase I feedback regulatory protein (GFRP). We here report and characterize a novel, Cre-Lox-driven, transgenic mouse model that overexpresses Gfrp. Results: Compared to control littermates, transgenic mice exhibited high transgene copy numbers, increased Gfrp mRNA and GFRP expression, enhanced GFRP-GTPCH1 interaction, reduced BH4 levels, and low glutathione (GSH) levels and differential mitochondrial bioenergetic profiles. After exposure to total body irradiation, transgenic mice showed decreased BH4/7,8-dihydrobiopterin ratios, increased vascular oxidative stress, and reduced white blood cell counts compared with controls. Innovation and Conclusion: This novel Gfrp knock-in transgenic mouse model allows elucidation of the role of GFRP in the regulation of BH4 biosynthesis. This model is a valuable tool to study the involvement of BH4 in whole body and tissue-specific radiation responses and other conditions associated with oxidative stress.