Kinetics Of DNA Double Strand Breaks Repair In Deinococcus Radiodurans

Objective:Deinococcus radiodurans(DR)is one of the most radioresistant organisms in the world.Ionizing radiation can induce a large number of double strand breaks(DSBs).RecA is a key enzyme in the process of homologous recombination repair of DSB by D.radiodurans.The purpose of this study is to detect the location and quantitative information of fluorescent labeled RecA,describing the reaction and diffusion process mathematically,establishing a dynamic model of RecA recruitment suitable for D.radiodurans,and finding out the reasons for the high efficiency of D.radiodurans in repairing DSB,which is of great significance to reveal the speed limiting steps in the repair process.Method:1.Using D.radiodurans genome and pUCm-T-syfp2 plasmid as templates,syfp2 with Nde I and not I restriction sites and recA with not I and Bam HI restriction sites were amplified by PCR,and then the pRADK-syfp2-recA vector was constructed by three segments ligation with T4 ligase after being digested with pRADK plasmid.pRADK-syfp2-recA was transformed into D.radiodurans by Ca Cl₂method to observe the non quantitative behavior of RecA.2.Large scale amplification of pRADK-syfp2-recA vector,amplification of thpR gene with Asc I at the end by D.radiodurans genomic PCR,amplification of Kan resistance gene with Asc I and Nde I at the end by pRADK as template PCR,construction of thpR-kan R-syfp2-recA linear DNA molecule by enzyme digestion and ligation,and transformation into DR by Ca Cl₂method.Stable transgenic D.radiodurans were obtained to observe the quantitative behavior of RecA.3.After irradiated on the ice,transformed DR were placed on the confocal glass slide,maintained at 30?,and the fluorescence photos were taken under lsm880 confocal laser microscope before and after irradiation to calculate the real-time fluorescence intensity.4.Using the continuous external pressure reaction chain model and other equations to fit the kinetic parameters,and comparing with the open data set,we can obtain new discoveries of DNA repair process.Result:1.The length of 1000bp recA and 750bp syfp2 fragments were obtained by PCR reaction,and the recombinant vector pRADK-syfp2-recA was obtained by three-stage ligation.The sequencing results showed that the constructed sequence was consistent with the template sequence without base mutation.pRADK-syfp2-recA was transformed into D.radiodurans.The positive clones were screened by amp,and the transformed Dr with stable expression of the target fusion protein was obtained.2.After 1600Gy?irradiation on ice,the continuous recruitment kinetics of RecA was observed under laser confocal microscope.Unexpectedly,it was observed that RecA was mostly bound to the cell membrane when it was not irradiated.After being stimulated by radiation,RecA migrated to the DNA region.With the progress of repair,RecA was slowly removed from the DNA and re bound to the cell membrane.3.The results of non quantitative behavior analysis showed that the elimination kinetics of RecA showed a first-order exponential decay,and the goodness of fit R²=0.998928.The third-order CRC model is used to fit the convolution radiation square wave signal to obtain the specific characteristic parameters.The results of RecA recruitment dynamics were in good agreement with the model prediction,R²=0.9853.4.Compared with the published data of human Rad51,the elimination rate constant of RecA(DR)is 265 times of Rad51(Homo sapiens),but the recruitment rate of RecA(DR)is lower than Rad51(Homo sapiens).5.A 700 bp thpR gene and an 800 bp kan R fragment were obtained by PCR reaction.The thpR-kan R-syfp2-recA linear DNA molecule was obtained by three-step ligation with T4 ligase and directly used for transfection.6.After 1600Gy?irradiation on ice,the expression kinetics of RecA was observed under confocal laser microscope after irradiation on ice.It was observed that the expression of RecA increased 1.7 times after irradiation,and reached the peak within 1 h.7.The results of quantitative behavior analysis showed that the expression kinetics of RecA showed the characteristics of expression activation function,and the goodness of fit R²=0.999851.Compared with the published microarray expression parameters,the RecA expression kinetic parameters are in good agreement.Conclusion1.In this study,two kinds of D.radiodurans expressing SYFP2-RecA fusion protein were constructed by transient transfection and stable transfection respectively,which can detect RecA kinetics under laser confocal microscope,and provide a feasible method for kinetic study.2.Based on the experimental data of expression and migration behavior of RecA,a dynamic model of RecA recruitment behavior of D.radiodurans was established by using the third-order CRC model to convolute the radiation square wave signal.The model can be used not only to predict the repair process of D.radiodurans in response to instantaneous radiation,but also to predict the dynamics of the repair process while radiation,It greatly improves our understanding of the driving factors of radiation reactions across time scales.3.Compared with human Rad51 parameters,RecA(DR)was eliminated more quickly from DNA region at the same scale,but the difference of recruitment rate was not significant,which indicated that the advantage of D.radiodurans in DSB repair was not through rapid recruitment of RecA,but in the efficient enzymatic reaction before and after binding,including terminal processing rate,DNA binding rate and DNA binding rate Among them,RecA mediated downstream processes play an important role in the rapid repair of D.radiodurans.4.Comparing the dynamic fluorescence parameters of RecA(DR)expression with those of gene chip,the change of fluorescence intensity with time was almost the same as that of RecA expression measured by gene chip,and the DSB repair rate constant of D.radiodurans was consistent at different doses.