Assessment of DNA-protein crosslink levels in HEK293T cells in first minutes after irradiation using comet assay.

Background: The role of DNA-protein crosslinks (DPCs) in radiation-induced damage effects in human cells and the dynamics of their repair following sublethal irradiation remain poorly understood. Unrepaired DPCs can lead to genome instability. The knowledge of biological impacts of ionizing radiation-induced DPCs (IR-induced DPCs) is essential for development of cancer treatments and approaches to radioresistance regulation.

Purpose: Purpose of the study was to estimate the effects of DPCs formation on radiation-induced DNA damage levels and DNA repair rate detected by comet assay in HEK293T cells.

Materials and methods: HEK293T cells were gamma-irradiated at doses 2, 4, 6, and 12 Gy. The alkaline comet assay was performed; distributions of cells by % DNA in comet tail were analyzed. Cells were fixed 1, 3, 5, 10, and 30 min after irradiation to estimate DNA repair dynamic. The proteinase K treatment and irradiation at 4 °C and 37° was used to estimate the contribution of DPCs formation and repair to the distribution of cells by DNA mobility.

Results and conclusions: Increased proportion of nucleoids with low electrophoretic mobility was shown in the first minutes after the γ-irradiation at 2 Gy. Proteinase K treatment of samples eliminated this effect, supporting our hypothesis of the DPCs being the main cause of this phenomenon. DPCs continued forming for several minutes after irradiation (increase of low-mobility nucleoids was observed up to 10 min after the irradiation); their repair rate was quite high (repaired within the first 30 min), comparable to the repair rate of single strand DNA breaks.