title: Effects of O-GlcNAcylation on radiation-induced DNA double-strand break repair
Creators: name: Averbek, Sera
Publisher: name: UNSPECIFIED
Year: 2021

description: DNA integrity is continuously threatened by various hazards such as cytotoxic chemicals, radiation or metabolic reactions, which potentially inflict DNA damage including DNA double-strand breaks (DSBs), the most harmful type of DNA damage. To maintain genome integrity, damaged DNA must be repaired accurately to prevent genomic instability that leads to diseases including cancer. Post translational modifications (PTMs) such as phosphorylation, ubiquitination or acetylation are essential to orchestrate DNA damage-response and repair mechanisms. Recent studies revealed an involvement of O-linked-N-acetylglucosaminylation (O-GlcNAcylation) in DNA damage response, which is an abundant, dynamic and reversible PTM of cellular proteins and very sensitive to environmental nutrient supply. O-GlcNAcylation occurs at amino acid Ser or Thr residues, which are also a target of phosphorylation. Therefore, O-GlcNAcylation and phosphorylation are suggested to compete. The aim of the study was to investigate different aspects of the regulatory role of O-GlcNAcylation on DNA-damage response in dependence of damage complexity and cell-cycle phase. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) inhibitors were used in order to change cellular O-GlcNAcylation level. DNA damage of different complexity was induced with low (X-rays) or high LET (linear energy transfer) radiation (C-, Fe- and He-ion) in selected human cell lines. Different approaches were used including γH2AX-foci assay, live-cell microscopy, Fluorescence Lifetime Microscopy (FLIM), westernblot analysis and protein-immunoprecipitation to detect DSB rejoining, accumulation of NBS1 at DNA damage site, chromatin states and O-GlcNAcylation of repair relevant factors upon irradiation respectively. First, induction of O-GlcNAcylation at the DSB site was examined with fluorescence co-localization analysis of 53BP1 and O-GlcNAc. They showed a radiation induced increase of O-GlcNAcylation within the nuclear compartment after X-ray or heavy-ion irradiation; specific O-GlcNAcylation at DSB sites however, was observed after heavy-ion irradiation only. The influence of O-GlcNAcylation on DSB repair was examined after X-ray and charged particles irradiation. It revealed that O-GlcNAcylation promotes DSB repair independent of DNA-damage complexity. Promotion of DSB repair occurs by stimulating homologous recombination (HR) and potentially an additional repair mechanism, which was revealed by studying repair of radiation induced DSBs in dependence of an siRNA-mediated knockdown of HR factor RAD51 together with modulating O-GlcNAcylation. Live-cell experiments suggested that inhibition of OGT disturbs the accumulation of NBS1 to X-ray induced DSBs. Moreover, MDC1 was confirmed to be O-GlcNAcylated, and HR factors CtIP and BRCA1 were shown to be modulated by O-GlcNAcylation upon irradiation. OGT and OGA activity also regulate chromatin compaction states that are proposed to be an important determinant of DSB repair pathway choice. Collectively, the data demonstrated multiple roles of O-GlcNAcylation in the repair of radiation induced DSBs concluding that O-GlcNAcylation is an important PTM to maintain genomic stability.