Browsing by Subject "Endodeoxyribonucleases"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair
    (Public Library of Science (PloS), 2015-12) Wu, Yuehan; Lee, Suk-Hee; Williamson, Elizabeth A.; Reinert, Brian L.; Cho, Ju Hwan; Xia, Fen; Jaiswal, Aruna Shanker; Srinivasan, Gayathri; Patel, Bhavita; Brantley, Alexis; Zhou, Daohong; Shao, Lijian; Pathak, Rupak; Hauer-Jensen, Martin; Singh, Sudha; Kong, Kimi; Wu, Xaiohua; Kim, Hyun-Suk; Beissbarth, Timothy; Gaedcke, Jochen; Burma, Sandeep; Nickoloff, Jac A.; Hromas, Robert A.; Department of Biochemistry and Molecular Biology, IU School of Medicine
    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.
  • Thumbnail Image
    Item
    STING Contributes to Abnormal Bone Formation Induced by Deficiency of DNase II in Mice
    (Wiley, 2017-02) Baum, Rebecca; Sharma, Shruti; Organ, Jason M.; Jakobs, Christopher; Hornung, Veit; Burr, David B.; Marshak-Rothstein, Ann; Fitzgerald, Katherine A.; Gravallese, Ellen M.; Anatomy and Cell Biology, School of Medicine
    OBJECTIVE: Cytosolic DNA sensors detect microbial DNA and promote type I interferon (IFN) and proinflammatory cytokine production through the adaptor stimulator of IFN genes (STING) to resolve infection. Endogenous DNA also engages the STING pathway, contributing to autoimmune disease. This study sought to identify the role of STING in regulating bone formation and to define the bone phenotype and its pathophysiologic mechanisms in arthritic mice double deficient in DNase II and IFN-α/β/ω receptor (IFNAR) (DNase II-/- /IFNAR-/- double-knockout [DKO] mice) compared with controls. METHODS: Bone parameters were evaluated by micro-computed tomography and histomorphometry in DKO mice in comparison with mice triple deficient in STING, DNase II, and IFNAR and control mice. Cell culture techniques were employed to determine the parameters of osteoclast and osteoblast differentiation and function. NanoString and Affymetrix array analyses were performed to identify factors promoting ectopic bone formation. RESULTS: Despite the expression of proinflammatory cytokines that would be expected to induce bone loss in the skeleton of DKO mice, the results, paradoxically, demonstrated an accumulation of bone in the long bones and spleens, sites of erythropoiesis and robust DNA accrual. In addition, factors promoting osteoblast recruitment and function were induced. Deficiency of STING significantly inhibited bone accrual. CONCLUSION: These data reveal a novel role for cytosolic DNA sensor pathways in bone in the setting of autoimmune disease. The results demonstrate the requirement of an intact STING pathway for bone formation in this model, a finding that may have relevance to autoimmune diseases in which DNA plays a pathogenic role. Identification of pathways linking innate immunity and bone could reveal novel targets for the treatment of bone abnormalities in human autoimmune diseases.