Deep-sea Extreme Thermophilic Archaea Endonuclease That Drives The Repair Of Hypoxanthine In DNA

Hypoxanthine(I)is a common damaged base in DNA,which is mainly derived from adenine deamination,and is mutagenic.High temperatures accelerate the rate at which adenine deamination forms I,suggesting that genomic DNA stability of hyperthermophilic archaea is severely challenged.Endonucleases are key enzymes that participate in repair of I in DNA.However,the mechanism of repair of I in DNA in hyperthermophilic archaea is still unclear.Both archaeon Thermococcus barophilus and Thermococcus gammatolerans were isolated from deep-sea hydrothermal vents.Their optimal growth temperature is about 85?.Genomic DNA sequencing of these two archaea has been completed,encoding both EndoV and NucS.In this thesis,Two endonucleases(Tba EndoV and Tga NucS)were used as the research objects,and the biochemical properties of cleaving I-containing DNA by endonucleases were studied,and the catalytic mechanism of Tga NucS was preliminarily discussed.The first part of this thesis mainly studied the gene cloning,induction expression and purification of Tba EndoV and Tga NucS.First,the Tba EndoV and Tga NucS genes were amplified by PCR and further cloned into the pET-30a(+)expression vector.The expression vector containing the targeted genes was transformed into the competent cells to express the strain by heat shock.Expression of the targeted protein was expressed by addition of IPTG(Isopropyl ?-D-Thiogalactoside).The Tba EndoV and Tga NucS proteins were then obtained by heat treatment and affinity purification of a Ni columnBiochemical charcaterization of cleaving I-containing DNA by Tba EndoV was studied in the second part of this thesis.In the study,we found that Tba EndoV didn't cleave normal and uracil-containing DNA,but could cleave I-containing DNA.The optimal temperature for the enzyme to cleave I-containing ssDNA was 50 to 90?.After heating the enzyme at 100? for 90 min,the efficiency of cleaving I-containing ssDNA by the enzyme was still 90%,indicating that the enzyme was a thermostable endonuclease.In addition,we found that the optimal pH for Tba EndoV to cut I-containing ssDNA was from 7.5 to 9.0.a divalent metal ion was required for the enzyme to cleave I-containing ssDNA.In the presence of Ni2+,Mg2+or Mn2+,Tba EndoV could cleave I-containing ssDNA with the efficiency in the order:Mn2+>Mg2+>Ni2+.Tba EndoV was able to tolerate 300 mM NaCl,but its activity was inhibited by higher concentrations of NaCl(>300 mM).Further studies showed that Tba EndoV was more capable of cleavage of I-containing ssDNA than dsDNA at the same temperature.The results of DNA binding experiments showed that Tba EndoV had stronger ability to bind ssDNA than dsDNA.Therefore,the enzyme was more likely to recognize I-containing ssDNA and display its cleavage activity.The third part of this thesis is about the study of the biochemical properties of cleaving I-containing DNA by Tga NucS,In this study,we found that Tga NucS was able to cleave I-containing ssDNA at 55? and cleave I-containing dsDNA at 80?.In addition,the optimal reaction temperature for cutting dsDNA by Tga NucS was 75?80?,and the optimal reaction pH was pH 7.0?9.0.A divalent metal ion was needed for the enzyme to cleave dsDNA,of which Mn2+ and Mg2+ are the most suitable metal ions.In addition,high salt inhibits Tga NucS from cleaving dsDNA.Further studies found that Tga NucS is similar to restriction endonuclease,and it cleaved two strands of DNA,where its cleavage position was the second phosphodiester bond at the 5' end of hypoxanthine and the third phosphodiester bond at the 5' end of the corresponding base T in the complementary template strand.Therefore,the cleavage product of this enzyme was short dsDNA with a 4'end overhang at the 5' end.Through the re-ligation experiment of the cleavage product of Tga NucS,we found that the cleaved products possess 3' OH and 5' P.Compared with the wild-type Tga NucS protein,the D163A mutant protein was unable to cleave I-containing dsDNA,the E177A mutant protein cleaved I-containing dsDNA with an efficiency of only 18%.Thus,amino acid residue D163 is one of the key active sites of the enzyme.The results of DNA binding experiments showed that the efficiency of binding of D163A mutant protein to I-containing dsDNA was 68%,which was lower than that of wild-type Tga NucS protein.In addition,the efficiency of binding of the E177A mutant protein to the I-containing DNA was similar to that of the wild-type protein.Therefore,D163 is involved in both cleavage and binding of dsDNA,while E177 is not involved in the binding of dsDNA,but partially partcipates in cleaving dsDNA.In this thesis,the biochemical properties of cleaving I-containing DNA by Tba EndoV and Tga NucS were revealed,and the catalytic mechanism of Tga NucS endonuclease was preliminary explored.The research findings of this thesis present important clues for clarifying repair of hypoxanthine in DNA of hyperthermophilic archaea,and also provide a basis for revealing the mechanism of DNA repair in hyperthermophilic archaea.