Biochemical characterization and mutational studies of the 8-oxoguanine DNA glycosylase from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans

Applied Microbiology and Biotechnology
Likui ZhangJianting Zheng

Abstract

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 m...Continue Reading

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Citations

Dec 2, 2020·Open Biology·Patricia Pérez-ArnaizThorsten Allers
Oct 30, 2020·Biomolecules·Craig J Marshall, Thomas J Santangelo
Jul 6, 2021·Applied Microbiology and Biotechnology·Likui ZhangPhilippe Oger

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Datasets Mentioned

BETA
ACS34155.1

Methods Mentioned

BETA
PCR
Protein Assay
glycosylase
electrophoresis
glycosylases

Software Mentioned

KaleidaGraph
SPSS Statistics
SPSS

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