Complex DNA Damage Induced by High Linear Energy Transfer Alpha-Particles and Protons Triggers a Specific Cellular DNA Damage Response

Abstract

PurposeTo investigate the precise mechanism of recognition and processing of ionizing radiation (IR)-induced complex DNA damage (CDD), where two or more DNA lesions are in close proximity, in cellular DNA which is packaged with histones to form chromatin.Methods and MaterialsHeLa and oropharyngeal squamous cell carcinoma (UMSCC74A and UMSCC6) cells were irradiated with high linear energy transfer (LET) α-particles or protons, versus low-LET protons and X rays. At various time points after irradiation, site-specific histone post-translational modifications were analyzed by quantitative Western blotting; DNA damage and repair were measured by different versions of the comet assay; and cell survival was determined using clonogenic assays.ResultsSite-specific histone post-translational modifications after low- and high-LET radiation, particularly proton irradiation, were screened, aiming to identify those responsive to CDD. We demonstrate that histone H2B ubiquitylated on lysine 120 (H2Bub) is specifically induced several hours after irradiation in response to high-LET α-particles and protons but not by low-LET protons or X rays/γ-radiation. This is associated with increased levels of CDD, which contributes to decreased cell survival. We further discovered that modulation of H2Bub is under the control of two E3 ubiquitin ligases, MSL2 and RNF20/RNF40 complex, whose depletion leads to defective processing and further persistence of CDD, and to additional decreased cell survival after irradiation.ConclusionThis study demonstrates that the signaling and repair of CDD, particularly induced by high-LET IR is co-ordinated through the specific induction of H2Bub catalyzed by MSL2 and RNF20/40, a mechanism that contributes significantly to cell survival after irradiation.

The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

“…We performed neutral DNA comet assays to directly quantify amounts of genomic DSBs (Fig A; Nickson et al , ; Carter et al , ). Depletion of ATX3 with two different siRNAs significantly increased the level of DSBs even in unchallenged conditions.…”

“…HeLa cells were treated with 20 nM non‐targeting control siRNA (Qiagen) or ATXN3 siRNA for 48 h, trypsinised and irradiated in suspension at 4 Gy using a CellRad X‐ray irradiator (Faxitron). Cells were embedded in agarose on a microscope slide, incubated for up to 4 h in a humidified chamber and levels of DNA double‐strand breaks measured using the neutral comet assay, as previously described (Nickson et al , ; Carter et al , ).…”

Characterisation of deubiquitylating enzymes in the cellular response to high-LET ionising radiation and complex DNA damage

“…This has been shown indirectly by demonstrating that protons with increasing LET cause reduced cell survival (6,7) and increases in persistent DNA DSBs as revealed by 53BP1 foci (8). However, recent data from our laboratory has directly demonstrated using an enzyme modified neutral comet assay that low energy (relatively high-LET) protons generate significantly increased amounts of CDD compared to high energy (low-LET) protons or x-rays which persists for several hours postirradiation (9).…”

“…Irradiation sources are as previously described (9). Briefly, cells grown in 35 mm dishes were exposed to low-LET x-rays (100 kV) using a CellRad x-ray irradiation (Faxitron Bioptics, Tucson, USA).…”

“…We discovered that this is co-ordinated by the E3 ubiquitin ligases RNF20/40 and MSL2 that play important roles in the repair of CDD and in cell survival following high-LET protons. We postulated that this is a mechanism for enhancing CDD repair by promoting chromatin remodelling and/or actively recruiting DNA repair enzymes (9). Nevertheless this study has identified that ubiquitylation, particularly of histones, plays an important role in the cellular response to IR-induced CDD.…”