Goodarzi, Aaron A.Moore, Shaun2018-07-032018-07-032018-06-21Moore, S. (2018). CHD6 and the oxidative stress-induced DNA damage response (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/32051http://hdl.handle.net/1880/106829Oxidative stress-induced DNA damage is a threat to the health and survival of a cell. Oxidative DNA damage responses involve nucleosome displacement, exchange or removal by ATP-dependent chromatin remodeling enzymes to promote DNA repair and transcriptional events. The CHD6 (Chromodomain, Helicase, DNA-binding 6) chromatin remodeling enzyme has been identified as an interactor of NFE2-related factor 2 (NRF2), a key transcription factor in the oxidative stress response. Several human ataxias have linkage map regions that encompass the CHD6 gene locus (20q11.1-12), while catalytically-inactive CHD6 mutant mice exhibit motor coordination defects most consistent with a cerebellar neuron disorder. Here, I describe a role for CHD6 in the response to oxidative stress-induced DNA damage. CHD6 relocates rapidly to DNA damage caused by microirradiation or KillerRed-induced oxidative stress, but not to enzyme-induced DNA double strand breaks. CHD6 interacts with poly ADP-ribose (PAR), and retention at laser microirradiation-induced DNA damage sites is PAR polymerase-dependent and prolonged by PAR Glycohydrolase depletion. I have narrowed down the PAR-interaction region of CHD6 to the extreme N-terminus and, using laser microirradiation, I have demonstrated the importance of the double chromodomain and a putative DNA-binding domain for normal recruitment to DNA-damage tracks. CHD6 protein levels are stabilized following H2O2 exposure via suppressed proteolytic degradation. Ablation of CHD6 in A549 cells using CRISPR-Cas9 led to elevated reactive oxygen species levels, created an impaired antioxidant response and a reduced ability to survive or proliferate following chronic H2O2 exposure. CHD6-deleted cells displayed elevated γH2AX and 53BP1 foci, increased ATM auto-phosphorylation and a hypersensitive G2/M checkpoint after exposure to ionizing radiation or H2O2. In conclusion, my data identify CHD6 as a novel responder to oxidative stress-induced DNA damage.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Biology--MolecularBiochemistrydoctoral thesis10.11575/PRISM/32051