Complex DNA damage induced by high-LET a-particles and protons triggers a specific cellular DNA damage response

Purpose Complex DNA damage (CDD), where two or more DNA lesions are induced in close proximity, is a signature of ionising radiation (IR) that contributes significantly to cell killing due to the difficult nature of its repair. However the precise mechanism of recognition and processing of CDD in cellular DNA, where the DNA is wrapped around histone proteins to form chromatin, is currently unknown. Methods and Materials HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A and UMSCC6) cells were irradiated with high linear energy transfer (LET) -particles or protons, versus low-LET protons and x-rays. At various time points post-irradiation, site-specific histone post-translational modifications (PTMs) were analysed by quantitative Western blotting, DNA damage and repair was measured by different versions of the comet assay, and cell survival was determined using clonogenic assays. Results Site-specific histone PTMs following 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 post-irradiation in response to high-LET α-particles and protons, but not by low-LET protons or x-rays. This is associated with increased levels of CDD and 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 post-irradiation. Conclusion This study demonstrates that the signalling and repair of CDD, particularly induced by high-LET IR is co-ordinated through the specific induction of H2Bub catalysed by MSL2 and RNF20/40, a mechanism which contributes significantly to cell survival post-irradiation.