Studies On The Biochemical Characterization And Catalytic Mechanism Of A Uracil DNA Glycosylase From Deep-sea Hyperthermophilic And Piezophilic Archaeon Thermococcus Barophilus Ch5

Hyperthermophilic archaea mainly thrive in high temperature environments such as deep-sea hydrothermal vents,craters,and terrestrial hot springs.High temperature would increase the rates of deamination of bases in DNA and form damaged bases.Further copying these damaged bases before being repaired usually would cause gene mutation.Cytosine is deaminated to form uracil,which is the most common type of deamination in DNA.Before it is repaired,replication of uracil in DNA would cause a G:C to A:T mutation.Uracil DNA glycosylase?UDG?is the first key enzyme for cells to initiate a base excision repair pathway to repair uracil in DNA.UDG is widely distributed in bacteria,archaea,eukaryotes and some viruses.UDGs are divided into six families based on their specificity in recognizing substrates.Currently,the hyperthermophilic archaea whose genomes have been sequenced encode at least one UDG.The hyperthermophilic and piezophilic archaeon Thermococcus barophilus Ch5 was isolated from the deep-sea hydrothermal vent of the mid-Atlantic ridge?Logachev oil field chimney,3020 meters deep?.Its optimal growth temperature and pressure are 85? and 40 Mpa,respectively.Two UDGs?Tba UDG194 and Tba UDG247?are encoded in the genome of T.barophilus Ch5,and their corresponding genes are TbCh5v1₂287 and TbCh5v1₀629,respectively.In this thesis,we used Tba UDG247 as the research object to study its biochemical characterization and catalytic mechanism for removing uracil from DNA.The first part of this thesis mainly carried out the research by molecular biology technique on gene cloning,induced expression and purification of Tba UDG247 protein.First,PCR technology was used to amplify the gene of Tba UDG247,which was then connected to pET-3 0a?+?expression vector after enzyme digestion.The successful recombinant plasmid by sequencing was transformed into the expressing strain by heat shock method.Tba UDG247 protein was then purified by cell disruption,heat treatment and Ni column affinity methods.The second part of this thesis studied the biochemical characterization of Tba UDG247 on excising uracil from DNA.We found that the recombinant Tba UDG247 only can excise uracil from ssDNA and dsDNA at 65?,but not cleave normal ssDNA and dsDNA,and have the highest excising activity at 70-75?.The enzyme can remove uracil from DNA in the pH range of 4.0 to 11.0,and its optimal pH is 7.0-9.0.In addition,the activity of the enzyme is independent of divalent metal ions;however,both Zn2+and Cu2+completely inhibit the activity of the enzyme.High NaCl concentration also inhibits the activity of this enzyme.Furthermore,the enzyme removes uracil from DNA with the following efficiency order:U-ssDNA?U:G-dsDNA>U:T-dsDNA?U:C-dsDNA>U:A-dsDNA.The kinetic results showed that Tba UDG247 excises uracil from ssDNA and dsDNA at a similar rate.The DNA binding results showed that Tba UDG247 has a stronger affinity for U-ssDNA than for U-dsDNA.The third part of this thesis investigated the catalytic mechanism of Tba UDG247 to remove uracil from DNA.The crystal structure of the complex of the hyperthermophilic archaeon Sulfolobus tokodaii UDG with uracil demonstrates that the uracil is encapsulated with the amino acid residues E42,N82,H164 and F55 in this UDG,suggesting that these residues may be essential to recognize and excise uracil.Residues E118,N159,H126 and Y127 in Tba UDG247 correspond to residues E42,N82,H164 and F55 in S.tokodaii UDG,respectively,suggesting that these amino acid residues may be the key sites for Tba UDG247 to excise uracil from DNA.In order to confirm this hypothesis,we constructed the Tba UDG247 E118A,N159A,H126A and Y127A mutants by using site-directed mutation technology.According to the method of induced expression and purification of the wild-type Tba UDG247 protein,the above four mutant proteins were expressed and purified,and the reactions of uracil-containing DNA excising and binding by these four mutants were performed.We found that the E118A,N159A and H216A mutants completely abolished the excising activity and retained the weakened binding activity,suggesting that the amino acid residues E118,N159 and H216 in Tba UDG247 protein are essential for the removal and recognition of uracil.However,the Y127A mutant showed similar cleavage and binding efficiencies as observed in the wild-type protein,suggesting that the amino acid residue Y127 in Tba UDG247 may play a role in maintaining the protein conformation.The fourth part of this thesis discussed the characteristics of Tba UDG247 with a bifunctional activity.By aligning the amino acid sequence of Tba UDG247 with other hyperthermophilic archaea and bacteria UDGs,we found that the enzyme lacks some conserved motifs in the UDGs present in other hyperthermophilic archaea and bacteria.The phylogenetic tree analysis results showed that Tba UDG247 is only present in Thermococcus genus,while Tba UDG194 belonging to the fourth family is distributed in both Thermococcus genus and Pyrococcus genus,suggesting that Tba UDG247 is a novel uracil DNA glycosylase of Thermococcus genus.Further phylogenetic tree analysis results showed that Tba UDG247 is located in an independent branch between family ? and family ?,suggesting that the enzyme may be a novel family of glycosylase and have novel biochemical function.The results of DNA cleavage experiments demonstrated that Tba UDG247 can not only cleave the N-C glycosidic bond between uracil and deoxyribose in DNA,thereby removing the uracil to generate AP site,but also can further cleave the generated Advanced placement site.Thus,Tba UDG247 has both glycosylase activity and AP lyase activity.Hence,Tba UDG247 differs from all the reported monofunctional UDGs that possess only glycosylase activity,however,resembles a bifunctional DNA glycosylase that has both glycosylase activity and AP lyase activity.In this thesis,we discovered that Tba UDG247 is a novel bifunctional uracil DNA glycosylase,revealed the biochemical properties of the enzyme to excise uracil from DNA,and clarified the key amino acid residues in the enzyme to excise uracil from DNA.Our research results on the biochemical properties,catalytic mechanism,and phylogenetic analysis of Tba UDG247 provide several important clues to clarify the base excision repair pathway for repair of uracil in the DNA in hyperthermophilic archaea.In addition,the discovery of the novel function of Tba UDG247 revealed in this thesis provides a basis for clarifying the evolutionary relationship of uracil DNA glycosylase,and also provides important evidence for the evolution of mono-and bi-functional DNA glycosylases.Furthermore,the Tba UDG247 studied in this thesis is higly thermostable,suggesting that this enzyme has potential application prospects in the field of molecular biology technology research and has enriched the extreme enzyme resources of the deep-sea thermophilic environments...