Shedding Light on the Biological Effects of Ionising Radiation on DNA Using Advanced Optical Microscopy
Detrimental effects of ionising radiation (IR) stem from its unique ability to produce clustered DNA damage (CDD) and double-strand breaks (DSBs). CDD consists of two or more lesions in close proximity, including DSBs, single-strand breaks and/or base damages. CDD complexity increases with rising linear energy transfer (LET) of radiation, while repairability decreases. This makes high-LET IR more biologically effective than low-LET. Understanding DNA repair mechanisms and associated signalling with radiation quality, may be useful in assessing exposure risks and also optimising radiotherapy. Despite this, CDD recognition and processing induced by different radiation qualities are currently unclear. In this study, kinetics, morphology and localisation of DSB (gammaH2AX) and non-DSB (OGG1) clusters were measured at CDD sites using immunofluorescence and confocal microscopy (CM). Slower repair kinetics were observed for high-LET alpha-particles compared to low-LET gamma-rays. Additionally, PARP inhibitor Olaparib in combination with IR resulted in increased foci persistence. Although Ku70/Ku80 are known as major effectors in DSB repair and genome integrity, their interaction in relation to DSB repair has not been detected in living cells. Using GFP technology and advanced imaging, Ku70-80 interaction was shown for the first time in live cells, with the Ku heterodimer pre-formed in the absence of DNA damage. Most foci studies are performed using standard CM, but the resulting data is limited by their resolution. Emerging evidence suggests these foci are sub-divided in structural domains. Furthermore, there are discrepancies in DSB foci yields among different methods. To assess this, super-resolution microscopy was employed to compare foci kinetics and structure with CM. CM underestimated the number of X-ray-induced gammaH2AX and 53BP1 foci by a factor of 2-5 and overestimated their colocalisation. Therefore, superresolution is not only useful in investigating foci structure, but will provide significant improvements in identifying the spatial distribution of correlated damage sites along high-LET tracks.
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