The Repair Kinetics Of XRCC1 In DNA Lesions Induced By High-LET Heavy Ion Irradiation

High-energy heavy ions can effectively kill deep-seated tumors,and reduce the damage to normal tissues due to the favorable depth-dose distribution,which has obvious advantages in tumor treatment.On the other hand,the high biological effectiveness of heavy ions in inducing late effects poses a serious threat to manned space exploration.The responses of cells to dense damage induced by heavy ion irradiation,especially the DNA damage response（DDR）,are quite different from those to the sparse damage.DNA damage response is a highly coordinated process.The involvement of the factors in the DNA damage response largely depends on their biochemical reactions with each other and with the chromatin.Scaffold protein is considered to play a central role in coordinating the recruitment/dissociation of factors involved in DNA damage response.It stabilizes DNA damage and its surrounding environment,provides binding sites for repair factors,and promotes the complete repair process.The online live-cell imaging system based on the high energy microbeam facility at the Institute of Modern Physics,Chinese Academy of Sciences,was used to investigate the response of the scaffold protein X-ray repair cross complementary protein 1（XRCC1）at the localized DNA damage in high-LET heavy ion irradiated human fibrosarcoma cell HT1080（expressing XRCC1 tagged RFP）.Moreover,the nano resolution structure of the repair foci of XRCC1 and other proteins in human fibrosarcoma HT1080 cell after heavy ion irradiation was also observed by stochastic optical reconstruction microscopy（STORM）.The main conclusions are as follows:1)XRCC1 was recruited to the DNA damage with rapid reponse in a poly ADP-ribose polymerase（PARP）dependent manner.The recruitment/dissociation kinetics of XRCC1 well fitted the consecutive reaction model.2)We introduced the consecutive reaction model to analyze the recruitment and dissociation process of DNA damage response factors.The kinetics of XRCC1 were quantitatively analyzed by the recruitment rate constant k₁ and dissociation rate constant k₂ of the model.3)The fractionated irradiation of the same cells resulted in accelerated dissociation of XRCC1 at the previous damage sites,while the dissociated XRCC1 immediately recycled with a higher recruitment efficiency,revealing the XRCC1’s new rescue mechanism and its high turnover in DNA damage response.4)When the linear energy transfer（LET）of irradiated ions was higher,the recruitment rate of XRCC1 was faster and the dissociation rate was slower.The XRCC1 recruitment was faster and dissociation was slower in the G₂ phase than those in the G₁ phase.However,the number of ion hits had little effect on XRCC1’s kinetics.The higher density of DNA damage provided more binding sites for XRCC1,which may be more conducive to the recruitment and retention of XRCC1.5)The super-resolution imaging of the repair factory induced by heavy ion irradiation showed that the foci of XRCC1 and PARP-1 were small circles,while the foci of 53BP1 showed curved slender strips.Both co-localization and anti co-localization of XRCC1 and 53BP1 were found,which may reflect their cooperation and competition in the DNA damage repair.We introduced the consecutive reaction model to quantitatively analyze the kinetics of XRCC1 at DNA damage sites induced by high-LET heavy ion irradiation,and investigated the nano resolution structure of the DNA damage repair foci.The obtained results could benefit our understanding of the biochemical mechanism in the DNA damage response,providing a useful reference for heavy ion radiation therapy and space radiation protection.