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Cloning Expression and Biochemical Characterization of Uracil-dna Glycosylase from P. Furiosus

Author: LinLiBo
Tutor: LiuJianHua;XiaoHuaSheng
School: Shanghai Jiaotong University
Course: Biological Engineering
Keywords: P. furiosus Base excision repair (BEP) Uracil-DNA glycosylase Deamination damage
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 28
Quote: 0
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Abstract


Hyperthermophiles live at an environment of temperatures higher than 85°C or even over 110°C. High temperature results in a high potential of deamination of cytimidine to uracil, and adenine to hypoxanthine, which puts hyperthermophiles in big danger. Deamination is the process whereby the amino group of a protein is being separated from the acidic group, yet it can damage the DNA. If uracil and hypoxanthine are not repaired before genome replication, mutations will generate after next round replication. Hyperthermophiles develop several strategies for confronting uracil with damage. First, the dUTPase hydrolyzes the harmful dUTP. Second, various UDGs remove uracil base from DNA and complete the base excision repairing with other proteins. Base excision repair is completed by co-function of several proteins. First, the DNA glycosylase excises damaged bases from DNA and generate the abasic site (AP site). Second, the AP endonuclease cleaves the DNA at AP site. Third, the DNA polymerase incorporates one or several correct deoxyribonucleotide(s) and DNA ligase seals the nick.To illustrate the mechanism of deamination, the udg gene which coding uracil DNA glycosylase (UDG) of the hyperthermophilic archaea P. furiosus was cloned and characterized. P. furiosus UDG (PfUDG) has high sequence similarity to the family IV and V UDGs. The excision efficiency of uracil in DNA is as follows: U/T≈U/C > U/G≈U/AP≈U/- > U/U≈U/I≈U/A. The optimal temperature and pH value for uracil excision are 70°C and 9.0, respectively. The removal of dU was inhibited by the divalent ions of Mg, Ca, Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The phosphorothioates near uracil strongly inhibit the excision of uracil by PfUDG. Interestingly, pfu DNA polymerase, which tightly binds the uracil-carrying oligonucleotide, does not inhibit the excision by PfUDG, suggesting PfUDG in vivo function as the repair enzyme to excise uracil damage in genome.

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