Biochemical characterization and mutational studies of the 8-oxoguanine DNA glycosylase from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans
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8-oxoguanine (GO) is a major lesion found in DNA that arises from guanine oxidation. The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes an archaeal GO DNA glycosylase (Tg-AGOG). Here, we characterized biochemically Tg-AGOG and probed its GO removal mechanism by mutational studies. Tg-AGOG can remove GO from DNA at high temperature through a β-elimination reaction. The enzyme displays an optimal temperature, ca.85–95 °C, and an optimal pH, ca.7.0–8.5. In addition, Tg-AGOG activity is independent on a divalent metal ion. However, both Co2+ and Cu2+ inhibit its activity. The enzyme activity is also inhibited by NaCl. Furthermore, Tg-AGOG specifically cleaves GO-containing dsDNA in the order: GO:C, GO:T, GO:A, and GO:G. Moreover, the temperature dependence of cleavage rates of the enzyme was determined, and from this, the activation energy for GO removal from DNA was first estimated to be 16.9 ± 0.9 kcal/mol. In comparison with the wild-type Tg-AGOG, the R197A mutant has a reduced cleavage activity for GO-containing DNA, whereas both the P193A and F167A mutants exhibit similar cleavage activities for GO-containing DNA. While the mutations of P193 and F167 to Ala lead to increased binding, the mutation of R197 to Ala had no significant effect on binding. These observations suggest that residue R197 is involved in catalysis, and residues P193 and F167 are flexible for conformational change.
Keywords8oxoG-DNA glycosylase Base excision repair Thermococcus gammatolerans
We thank Prof. Fabrice Confalonieri at University of Paris-Sud for providing the genomic DNA of T. gammatolerans EJ3. We thank Qi Gan at Yangzhou University and Jing Zhao at Hebei Agricultural University for their assistance.
LZ and ZY designed experiments; YL, HS, and DZ performed experiments; LZ, ZY, PO, and JZ analyzed data; LZ, ZY, PO, and JZ wrote and revised the paper.
This work was supported by the Academic Leader of Middle and Young People of Yangzhou University Grant and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University (No. MMLKF18-05) to L.Z.; the practice innovation training program for Postgraudate students in Yangzhou University to H.S (No. 201711117059Y).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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