Establishment And Application Of A Novel Evaluation Method For DNA Single-strand Breaks Repair

DNA lesion and repair is an inherent feature of chromosome metabolism to overcome endogenous and exogenous damages.Among the different types of DNA damage,single-strand breaks(SSBs)are the most common,arising at a frequency of tens of thousands per cell per day.The single-strand break repair(SSBR)pathway is associated with cell aging and a variety of diseases,including cancer.However,the common methods to evaluate the SSBR capacity of cells are immunofluorescence and Western blot based on the difference in the SSBR-related protein expression and single-cell gel electrophoresis.Due to the strong compensatory capacity in cells,the difference in specific protein cannot represent the capacity of SSBR of the whole cell and SCGE is tedious and time-consuming.Therefore,it is necessary to establish a method to directly evaluate the SSBR capacity of cells.In this study,the SSBR ability of different cells and the effect of anti-aging drugs were evaluated based on the different fluorescence between pyrene excimer and monomer.First,we established and optimized the system.3’-pyrene-modified Oligo A and5’-pyrene-modified Oligo B with the complementary ends were synthesized.Pyrene monomer would produce blue fluorescence at 378 nm and 397 nm followed by being excited at 344 nm.After annealing,two pyrene monomers are close together to form an excimer,which produces green fluorescence at 488 nm.Two different fluorescence switches are used to evaluate the ability of SSBR,that is,when T4 PNK and T4 DNA ligase are added,the nick is repaired to maintain the excimer fluorescence.When T4 PNK or T4 DNA ligase is not present,one sequence is hydrolyzed,which turns off the excimer fluorescence and produces monomer fluorescence.By optimizing the concentration of Oligo,enzyme dosage,time,and temperature,the fluorescence value of the repaired system could reach about 10 times that of the unrepaired system.Subsequently,we applied the reporter system to directly evaluate the SSBR in different cells.We isolated and cultured various cells and obtained nucleoproteins.In mouse primary fetal fibroblasts,the excimer fluorescence was significantly higher than those of tumor cells and cell lines.In addition,the results showed that the fluorescence of the tumor cell system remained unchanged by supplementing DNA repair-related enzymes into the system of tumor cells.Finally,the fluorescence reporter system was applied to evaluate the effect of anti-aging drugs.It was found that SSBR capacity was significantly reduced during cell aging.However,in the cells supplemented with nicotinamide mononucleotide(NMN),niacin(NA),melatonin,and spermidine,the decline was effectively inhibited.In addition,we studied the aging-associated signaling pathways by Western blot and q PCR.In conclusion,we have successfully established a simple and feasible evaluation method for SSBR.Compared with fetal fibroblasts,the SSBR of tumor cells and cell lines can be ignored,and some factors in tumor cells might inhibit the SSBR of cells.At the same time,the proposed method was applied to the evaluation of anti-aging drugs,and it was found that NMN,NA,melatonin,and spermidine had obvious anti-aging effects,while the effect of vitamin C was not significant.Therefore,the system could be used to evaluate the effect of anti-aging drugs.Our strategy provides a new way to identify tumor cells,explore the mechanism of SSBR,and screen related drugs in the future.