Biochemical characterization and mutational studies of a thermostable uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Shi, Haoqiang; Gan, Qi; Jiang, Donghao; Wu, Yuqi; Yin, Youcheng; Hou, Haiyue; Chen, Hongxun; Xu, Yun; Miao, Li; Yang, Zhihui; Oger, Philippe; Zhang, Likui

doi:10.1016/j.ijbiomac.2019.05.073

Cited by 8 publications

(14 citation statements)

References 45 publications

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“…A divalent metal ion is not required for Tba UDG194 activity, which is congruent with Tba UDG247 (18) and other reported UDGs (21,22). Mn 2+ displays partial inhibition of Tba UDG194 activity, which has been observed in Tba UDG247 (18) and A. pernix UDG (22). Cu 2+ abolishes Tba UDG194 activity, but partially suppresses Tba UDG247 activity (18).…”

Section: Discussionsupporting

confidence: 89%

“…Mn 2+ displays partial inhibition of Tba UDG194 activity, which has been observed in Tba UDG247 (18) and A. pernix UDG (22). Cu 2+ abolishes Tba UDG194 activity, but partially suppresses Tba UDG247 activity (18). Furthermore, Tba UDG194 activity is partially inhibited by Zn 2+ , however, Tba UDG247 is inactive in the presence of Zn 2+ (18).…”

Section: Discussionmentioning

confidence: 77%

“…Furthermore, Tba UDG194 retains the compromised activity after heated at 95°C for 30 min. By contrast, Tba UDG247 abolishes its activity after treated with 95°C for 30 min (18), thereby suggesting that Tba UDG194 is more thermostable than Tba UDG247.…”

Section: Discussionmentioning

confidence: 94%

“…Among these six motifs, motifs B and F are ubiquitous in all families of UDGs. The conserved residues in motifs B and F are confirmed to be an active site center in UDGs (18).…”

Section: Tba Udg194 Is a Family IV Uracil Dna Glycosylasementioning

confidence: 84%

“…The hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 is one of the most extreme members of the Thermococcus genus, being hyperthermophilic (optimal temperature of 85 o C) and piezophilic (optimal pressure of 40 Mpa), and possesses two putative UDGs: Tba UDG194 and Tba UDG247, which are encoded by genes TbCh5v1_0629 and TbCh5v1_2287 (17), respectively. In our previous publication, we biochemically characterized Tba UDG247, demonstrating that this UDG can cleave uracil-containing ssDNA and dsDNA at high temperature (18). Sequence alignment shows that Tba UDG247 is only present in a few Thermococcus species, and is absent from all Pyrococcus.…”

Section: Introductionmentioning

confidence: 87%

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Characterization of a Family IV uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Gan

Shi

et al. 2020

International Journal of Biological Macromolecules

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The hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes two uracil DNA glycosylases (UDGs): Tba UDG247 and Tba UDG194. In our previous publication, we revealed biochemical characterization of Tba UDG247. Herein, we characterized biochemically Tba UDG194, which is a member of Family IV UDG, demonstrating that this enzyme has similar efficiencies for cleaving uracil-containing ssDNA and dsDNA. Compared with Tba UDG247, Tba UDG194 exhibits different biochemical characteristics. At >85 o C, >90 cleavage percentage was observed, suggesting that Tba UDG194 can remove uracil from DNA at the close physiological temperature of its host. Thus, the enzyme has been currently the most thermophilic glycosylase among all the reported UDGs. Furthermore, the optimal pH of the enzyme activity was estimated to be 10, which is higher than that of Tba UDG247. Similar to Tba UDG247, Tba UDG194 activity is independent on a divalent metal ion. Mn 2+ , Zn 2+ and Cu 2+ display inhibitory effect on the enzyme activity at varied degreed whereas Mg 2+ and Ca 2+ have no detectable effect on the enzyme activity. In addition, Tba UDG194 is a salt-tolerant enzyme that retains compromised activity at 600 mM NaCl. Furthermore, Tba UDG 194 displays the following substrate preference: U≈U/ G>U/T≈U/C>U/A. The Arrhenius activation energy was etsimated to be 20.1 ± 3.4 kcal/mol, theoretically representing the energy barrier for uracil removal from DNA by Tba UDG194. Overall, our observations suggest that Tba UDG194 might be involved in removal of uracil in DNA in Thermococcus cells.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 94%

“…Among these six motifs, motifs B and F are ubiquitous in all families of UDGs. The conserved residues in motifs B and F are confirmed to be an active site center in UDGs (18).…”

Section: Tba Udg194 Is a Family IV Uracil Dna Glycosylasementioning

confidence: 84%

Section: Introductionmentioning

confidence: 87%

See 3 more Smart Citations

Characterization of a Family IV uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Gan

Shi

et al. 2020

International Journal of Biological Macromolecules

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Identification of a novel bifunctional uracil DNA glycosylase from Thermococcus barophilus Ch5

Zhang

Jiang

Gan

et al. 2021

Appl Microbiol Biotechnol

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Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high-temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature.Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bond of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave APcontaining ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in the repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. The phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs.

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Characterization and application of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans

Zhang

Jiang

Shi

et al. 2020

International Journal of Biological Macromolecules

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Biochemical characterization and mutational studies of a thermostable uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Cited by 8 publications

References 45 publications

Characterization of a Family IV uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Characterization of a Family IV uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Identification of a novel bifunctional uracil DNA glycosylase from Thermococcus barophilus Ch5

Characterization and application of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans

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