Spatiotemporal Dynamics Of DNA Damage Repair Induced By Ionizing Radiation

Human DNA molecules exist in the nucleus in the form of chromatin through highly assembling.The dynamic of chromatin structure interacts with many biological processes in the nucleus.As genetic material in life,DNA double strand breaks(DSBs)are serious threat to genome stability,and the abnormality of DSBs repair would lead to gene mutation and cancer.Human health is often threatened by ionizing radiation in high radiation environment,radiation therapy and manned space activities.DNA damage response induced by ionizing radiation and DSBs repair process modify or recruit a variety of chromatin remodeling factors and repair proteins to form repair focus at the damage site.How can they be recruited quickly after damage? How do they move to damage sites through highly compressed chromatin? And how do they work at the damage sites? In this paper,we studied the kinetics of DSBs repair focus and the fine structure of chromatin.It is helpful to understand the mechanism of DNA damage repair and chromatin structure regulation after ionizing radiation,and provide scientific guidance for tumor radiotherapy and space radiation protection.First,based on immunofluorescence high-resolution microscopy and threedimensional image analysis,the dynamics of ?-H2 AX and 53BP1 proteins in HeLa cells irradiated by X-ray was studied.The results showed that the repair dynamics of DSBs were different in the focus number,intensity and volume of ?-H2 AX and 53BP1 in different irradiation doses.The focus number of ?-H2 AX and 53BP1 reached the maximum at 15 minutes after 0.2 Gy irradiation.After 1 Gy irradiation,?-H2 AX reached the maximum at 10 minutes,while 53BP1 reached the peak at 25 minutes.The time difference of the peak number of different protein focus reflected their time dependence in DNA damage response process.From the focus volume and intensity analysis,there were still some residual damages that could not be completely repaired after 24 hours of irradiation with 0.2 Gy and 1 Gy.The results showed that the three-dimensional high-resolution fluorescence analysis had a high sensitivity of focus analysis.With the help of super-resolution imaging technology,single molecule analysis and image reconstruction of DNA damage repair focus were carried out.Fine structure and distribution of ?-H2 AX,53BP1,MDC1 and XRCC1 at the damage sites were obtained.In the same damage site,MDC1/?-H2 AX and 53BP1 proteins were co-localization and partially independent distribution.The molecular number of 53BP1 protein was directly determined by that of ?-H2 AX.There was rare spatial co-location between 53BP1 and XRCC1.Little XRCC1 were located at 25 nm and 175 nm from the center of 53BP1,which might be the overlap of SSBs and DSBs.The spatial distribution and location differences of these repair proteins at nanoscale indicated that although they were in the same repair focus,their specific functions were different.The changes of chromatin structure in nucleus during DNA damage and repair were studied by ionizing radiation and super-resolution imaging analysis.The results showed that chromatin of non-irradiated samples were heterogenous high-and lowdensity areas accompanied by compact fibrous chromatin and small number of clusters.In the irradiated cells,sub-100 nm clusters were homogeneous distribution in nucleus.A large number of “star” shape clusters similar to chromatin were found in the single molecule reconstruction image of ?-H2 AX,53BP1 and MDC1 proteins,which might be the 30 nm chromatin fiber structure.Dual stain super-resolution imaging showed that repair process took place along the chromatin structure.Chromatin decondensation was key regulation for repair factors recruitment at the damage site.Meanwhile,the change of chromatin surface charge by H2 AX phosphorylation and the recruitment of chromatin remodeling factors might be the reasons for the phenomenon,which needed to be confirmed by further research.