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The Structural Character And Antioxidant Function Of Phosphorothioate DNA

Posted on:2018-07-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:T T WuFull Text:PDF
GTID:1360330590970496Subject:Biology
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Phosphorothioation is a new type of DNA modification,in which one non-bridging oxygen atom in phosphophate backbone was replaced by sulfur atom,with structural characterization of low frequency and stereoselectivity.This study focused on its antioxidant acitivity,spectroscopic characterization,and structural dynamics of phosphorothioated DNA.Antioxidant function of PT-DNA was firstly reported in 2012 by Wang et al,that is,PT-positive strain exhibited resisitance to oxidative stress in the presence of H2O2.However,some mechanistic problems were still unsolved: 1,the frequency of phosphorothioate DNA is in a range of 1/10000 to 1/1000 nt,so why the bacteria choose the rare phosphorothioation as an antioxidant "weapons" ?2.compared with the traditional surful reductant(cysteine and glutathione),is the phosphorothioate DNA has special advantages? 3,hydrogen phosphate was generated and led to the breakage of DNA strand under hydrogen peroxide oxidation;whether the by-product conflicted with PT-DNA antioxidant protection.Second,the traditional method of local PT detection was HPLC/MS or DNA sequencing,however,both of them could destroy DNA structure.At present,the detection methods including spectroscopy,nuclear magnetic resonance,and X-ray were difficult to reflect the low-frequent local structure of DNA and limited to detect short nucleotide.Thus,developing a convenient method for the PT modificatd plasmid DNA detection is particularly important.In addition,although a wealth of studies have been investigated on DNA backbone,less information of PT modified DNA structure was constructed and a completeatlas of the structure and dynamics of the PT-DNA,is so far lacking.In detail,this he dissertation includs four sections:In part I,the antioxidant activity of PT-DNA was investigated with comparision to traditional antioxidants,phosphorothioate compound presents an unexpectedly high capacity to quench hydroxyl radical.Oxidative product analysis by liquid chromatography-mass spectrometry and quantum mechanistic computation supported its unique anti-oxidant characteristic of the hydroxyl selectivity: phosphorothioate donates an electron to either hydroxyl radical or guanine radical derived from hydroxyl radical.When the concentrations of GSH and cysteine were in higher concentration,the antioxidant activity disappears and exhibits severe oxidative effects,that is,more reactive oxygen free radicals produced.However,this side effect does not occur in PS.In the process of PT-DNA skeleton cleavage in oxidation process,we analyzed the oxidation products of H2O2,ABTS·+ and GsA.ABTS·+ and H2O2 can oxidate GsA to GA.the PH generation needs two molecules of H2O2 with a higher reaction barrier,consistent with the HPLC-MS experimental observation in which the PH formation was highly dependent on the concentration of H2O2.High concentration of hydrogen peroxide could produce GHA which will cause the DNA strand breaks.It is also reasonable that eukaryote develops complicated karyotheca substructure tightly packing genetic materials away from mitochondria,while a prokaryote needs additional protector from oxidative metabolites.However,whether the PS-PO conversion cascades an in vivo ROS-signal to regulate PT-DNA expression in the ROS response needs further investigation.The novel mechanism of anti-ROS property of PT-DNA may be applicable in anti-tumor and anti-aging pharmaceutics.In part II,traditional method for phosphothioation identification is to use mass spectrometry or DNA sequencing,but both will destroy DNA sample.Whether a convenient and nondestructive detection method of Raman spectroscopy can be applied to detect phosphothioate DNA? Weused Raman spectroscopy to detect phosphorothioate modified and regular DNA in dinucleotide and plasmid DNA level.The results show that the peaks of stretching vibration of phosphorothioate bond were at 603 cm-1and 432 cm-1 and the minimum energy conformation of Sp tend to(anti,g-)and Rp was(g-,anti).However,the Raman spectra of the two configurations have no significant difference.Moreover,partial least squares discriminant analysis showed that the phosphothioation modified plasmid DNA is Gs A.The third part was the structural characteristics of PT modification by molecular simulation.In the previous study,we reported that the stiffness of the DNA backbone was enhanced by the phosphorothioate and the structural effect related to the sequence preference based on the 16 different dinuclotide.However,sequence dependence of the tetranucleotide,beyond dinucleotide level,showed that the impact of flapping base step was identified as a key factor of BI/BII transitions obtained from MD simulations.Thus,from the molecular dynamics,the molecular simulation of the Drew-Dcikerson dodecamer which the central base step of AATT was replaced by the whole conformational spaces of tetranucleotide(256 without modification and 256 for phosphorothioate modification),was used to investigate the effect of phosphorothioate DNA.We found that phosphorothioate modification caused the decrease of BI to BII transition.Previous studies found that canonical H-bonds associated with O3' and C6-H6/C8-H8 of the Y/R base at the 3' end of the central base step could reflect the BII occupancy.To further confirm our interpretation,we carried out quantum chemical calculations on PS and PO.The electron densities of O3' is-0.71 e for PS and-0.89 e for PO.As the decrease of charge in O3',C6-H6···O3' and C8-H8···O3' no longer come into close contact and the backbone undergoes significantly fewer transitions to the BII state.Thus,without the H-bond stabilizing interaction,the backbone cannot last in time in the BII state.In part IV,the interaction between the site of phosphorothioationmodification and methylase was studied by molecular simulation.The local structural instability of the protein could be induced by phosphorothioation modification;meanwhile,after Rp modification,the Rise and Roll angles would be increased,causing torsion of base plane,weakened ?-? interaction and decreased number of hydrogen bond,thereby resulting in the change of the recognition ability of local key amino acids and bases under the interaction between methylase and DNA.In addition,during the process of binding free energy analysis of DNA-methylase complexes,the electrostatic action energy provided the main driving force in the Van der Waals energy.Following Rp modification,there was a torsion of Arg137,which was difficult to produce the interaction with the base plane,thereby leading to the instability of the local structure.Therefore,the binding mode of methylase with its local DNA region was changed after Rp modification,which induced the decrease of enzyme activity and restricted the occurrence of methylation.In this paper,the molecular mechanism of resistance to oxidative damage was explored by combining the computational and experimental methods through the study of oxidation resistance.Furthermore,a direct and simple method was developed for detecting the site of phosphorothioation modification via Raman enhanced spectroscopy.Additionally,a theoretical model for the increase of BI after phosphorothioation modification was proposed for the first time by means of molecular dynamics simulation,and the mechanism of the reduced activity of the modified methylase was also then analyzed,all of which provided practical significance for subsequent experiment and theoretical research.
Keywords/Search Tags:DNA, phosphorothioation, antioxidant effect, Raman spectroscopy, molecular simulation
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