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The role of the histone variant H3.3 and its chaperones in the response to DNA damage 10.17863/CAM.84400 Walker, Lucy Alice Sale, Julian E H3.3 is a histone variant without a clear, single function. Unlike the canonical replicationassociated H3.1 and H3.2, it is present throughout the cell cycle and can be deposited into chromatin by two distinct and specific chaperone complexes: ATRX/DAXX and HIRA. H3.3 and its chaperones have been implicated in the response to and repair of various types of DNA damage. Furthermore, a supply of H3.3 has been shown to be required to maintain replication fork progression in the transformed avian cell line DT40, despite the availability during replication of canonical H3. In this thesis, I have examined the role of H3.3 and its chaperones in the response to UV induced DNA damage in a non-transformed, but immortalised, human cell system, the lymphoblastoid line TK6. Using CRISPR-Cas9 genome editing, I created H3.3, ATRX and HIRA knockout mutants and investigated their response to UV irradiation. h3.3 TK6 cells are not hypersensitive to acute DNA damage but exhibit persistence of DNA damage markers. h3.3 cells also exhibit a change in their cell cycle distribution after UV irradiation, with a G1 arrest after 24 hours. These observations support the hypothesis that there is a delay in resolving DNA damage in the absence of H3.3. The observed checkpoint activation is mediated by p53, as demonstrated by alleviation of the G1 arrest in h3.3/p53 double knockout cells. Surprisingly, in TK6, loss of H3.3 did not result in altered fork dynamics after UV irradiation. atrx cells phenocopy the h3.3 cells in these responses to acute DNA damage, likewise displaying a G1 accumulation 24 hours after UV irradiation, but hira cells do not. This suggests that the role of H3.3 in the response to UV induced DNA damage is likely mediated by ATRX-dependent deposition rather than deposition via a HIRA-dependent pathway. To further investigate the potential pathways in which H3.3 could be involved after UV induced damage, I present the results of a proteomic screen for H3.3 interactors and a CRISPR/Cas9 screen to determine genetic interactions of H3.3. in the response to DNA damage. Together, this work demonstrates a role for H3.3 in the response to DNA damage in nontransformed human cells and implicates deposition by its chaperone ATRX in this role. University of Cambridge 2021-09-30 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/336976 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/519ff1d6-77d6-4eb8-a42f-bf45693721c9/download 945e754ce2409847bd9b023de9448293 https://creativecommons.org/licenses/by/4.0/ H3.3 ATRX