PAR Modification And Acetylation Modification Of DNA-PKcs In Dna Damage Response

There are many environmental factors in nature,such as ultraviolet light,ionizing radiation and chemical agents,which can cause DNA damage,including DNA base damage,DNA adducts,DNA double-strand breaks,DNA single-strand breaks,DNA double strand breaks(DSB)is the most serious one,and is a major form of DNA damage included by ionizing radiation(IR).There are two major pathways of repairing DNA DSB: non-homologous end joining(NHEJ)and homologous recombination repair(HR).NHEJ repair is error-prone repair,HR is more accurate error free way of DSB repair.DNA-PKcs is a very important protein in DNA double-strand break repair.Its post-translational modifications play a decisive role in its activity.Currently,there are many kinds of protein post-translational modifications.Poly ADP ribosylation(PAR)modification and acetylation modification are two of them.These post-translational modifications play very important roles in chromatin remodeling,DNA repair,gene transcription,apoptosis and tumorigenesis.Moreover,phosphorylation also plays an important role in DNA repair.Objective:(1)Explore the PAR modification of DNA-PKcs and its effects on DNA-PKcs function associating with DNA damage repair.(2)Exploring the acetylation modification of DNA-PKcs.Methods:(1)After the cells being irradiated or without irradiation,Co-IP,immunofluorescence(IF),western blot,and flow cytometry were used to detect changes in PAR modification of DNA-PKcs and PAR modification level of DNA-PKcs in different phase of cell cycles;The effect of the poly ADP ribosylation(PARP)inhibitor Olaparib on DNA-PKcs and its impact on repair pathway selection;Cell clonogenic assays verified the radiosensitizing effect of Olaparib,The cell growth curve verified the toxic effects of DMSO,Olaparib,Olaparib+DNA-PKcs inhibitor NU7441 on cells;the expression of DNA-PKcs Ser2056 and ATM Ser1981 were examined after Olaparib and NU7441 treated alone or in combination;The expression of DNA-PKcs Ser2056 and ATM Ser1981 was also examined after the treatment of Olaparib and KU55933 alone or in combination.(2)The acetylation of DNA-PKcs was verified by co-immunoprecipitation(Co-IP)compared with the unirradiated group.The degree of acetylation at different times after irradiation was also verified;The degree of acetylation of DNA-PKcs in different phase of cell cycle was verified using the synchronized cells of different phase of cell cycle;potential acetylation sites for DNA-PKcs was predicted by using Tip60 sequences and functional properties in the ASEB website;DNA-PKcs acetylation was further detected in different truncation mutants;Mass spectrometry experiments was perfomed to search for possible acetylation sites for DNA-PKcs.Results:(1)After irradiation,PAR modification has been detected in DNA-PKcs,and the degree of PAR modification becomes higher as the irradiation dose increases;After treatment with Olaparib,the PAR modification was significantly reduced at a concentration of 10 ?M;Through the TDR cycle delay,the PAR modification is the more significant in the S phase;Western blotting and laser confocal microscopy showed that Olaparib increased the phosphorylation level of DNA-PKcs on S2056 site;After treatment of cells with Olaparib,the NHEJ repair pathway is activated while inhibition of HR repair;Olaparib increases the radiosensitivity of the cells;Olaparib treatment increases the ATM phosphorylation status.(2)DNA-PKcs can be acetylated and has the strongest acetylation in S phase.The acetylation modification is also enhanced with time after irradiation;The ASEB website predicted acetylation sites: K1917,K3650,K4004,K1870;Mass spectrometry results suggest that the acetylation site is K1870.Conclusion: DNA-PKcs can be parylated,and PAR modification increases with irradiation dose;and DNA-PKcs parylation is most obvious in S phase;DNA-PKcs and ATM activity was activated after treated with Olaparib;Olaparib treatment activates the NHEJ repair pathway while inhibited the HR pathway,and increases the radiation sensitivity of the cells.(2)DNA-PKcs can be acetylated,which increases with prolonged exposure time of radiation and is most pronounced in S phase;Possible modification sites for acetylation are: 1870,1917,3650,4004.