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

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 o C, and an optimal pH, ca.7.0-8.5. In addition, Tg-AGOG activity is independent on a divalent metal ion. However, both Co 2+ and Cu 2+ inhibit its activity. The enzyme activity is also inhibited by NaCl. Furthermore, Tg-AGOG specifically cleaves GOcontaining dsDNA in the order: GO:C, GO:T, GO:A and GO:G. In comparison with the wild-type Tg-AGOG, the R197A mutant has a reduced cleavage activity for GOcontaining 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.

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

“…Tba UDG194 is a classical family IV UDG member, possessing the conserved motifs present in other family IV members (Gan et al 2020). In contrast, Tba UDG247 lacks the conserved motifs found in family IV and family V UDG members, although being most closely related to family V UDGs (Shi et al 2019). Herein, we provide evidence that Tba UDG247 defines a novel family of bi-functional UDGs, which differs from all other reported UDGs.…”

“…Ch5 is both hyperthermophilic (an optimal temperature of 85 o C) and piezophilic (an optimal pressure of 40 MPa) (Marteinsson et al 1999). Our results have shown that both enzymes can remove uracil from DNA at high temperature (Shi et al 2019;Gan et al 2020). Tba UDG194 is a classical family IV UDG member, possessing the conserved motifs present in other family IV members (Gan et al 2020).…”

“…The sequences of complementary strand are 5′-TGA GGT GAT CGT TCG CTA CAY GTC GTC AGG ATT CCA GGC AGT TCG -3′ (Y: T, C or G). The Cy3-labeled oligonucleotide duplex was prepared as described previously (Shi et al 2019). Briefly, the Cy3 labeled deoxyoligonucleotides were annealed to the complementary deoxyoligonucleotides to prepare dsDNA in an annealing buffer (20 mM Tris-Cl pH 8.0 and 1 M NaCl).…”

“…Briefly, the Cy3 labeled deoxyoligonucleotides were annealed to the complementary deoxyoligonucleotides to prepare dsDNA in an annealing buffer (20 mM Tris-Cl pH 8.0 and 1 M NaCl). The annealing reactions were performed at 100 °C for 5 min and cooled slowly to room temperature at least 4 h. Tba UDG247 and an archaeal GO (8-oxoG) DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus gammatolerans (Tg-AGOG) were obtained as described previously (Shi et al 2019a;Zhang et al 2019). E. coli UDG was purchased from Thermo Scientific Company, USA.…”

Archaeal DNA Repair Mechanisms

“…Archaeal BER has been completely reconstituted in vitro ( Figure 7). The Ogg-subfamily archaeal GO glycosylase (AGOG) of Thermococcus kodakarensis [102] recognizes 8-oxo-guanine (8oxoG) modifications which result from the oxidation of guanine and acts as a bifunctional glycosylase to perform base excision and cleave the DNA backbone. The activity of AGOG-like BER enzymes leave a 1 nt gap with a 5 phosphate and 3 UA after recognition of a chemically modified base.…”