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.

Recent studies show that NucS endonucleases participate in mismatch repair in several archaea and bacteria. However, the function of archaeal NucS endonucleases has not been completely clarified. Here, we describe a NucS endonuclease from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans (Tga NucS) that can cleave uracil (U)-and hypoxanthine (I)-containing dsDNA at 80 o C. Biochemical evidence shows that the cleavage sites of the enzyme are at the second phosphodiester on the 5′-site of U or I, and at the third phosphodiester on the 5′-site of the opposite base of U or I, creating a double strand break with a 4-nt 5′overhang.The ends of the cleaved product of Tga NucS are ligatable, possessing 5′phosphate and 3′-hydroxyl termini, which can be utilized by DNA repair proteins or enzymes. Tga NucS displays a preference for U/G-and I/T-containing dsDNA over other pairs with U or I, suggesting that the enzyme is responsible for repair of U and I in DNA that arise from deamination. Biochemical characterization of cleaving U-and I-containing DNA by Tga NucS was also investigated. The DNA-binding results show that the enzyme exhibits a higher affinity for normal, U-and I-containing dsDNA than for normal, U-and I-containing ssDNA. Therefore, we present an alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease in hyperthermophilic archaea.

Uracil DNA glycosylases (UDGs) play an important role in removing uracil from DNA to initiate DNA base excision repair. Here, we first characterized biochemically a thermostable UDG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba UDG), and probed its mechanism by mutational analysis.The recombinant Tba UDG cleaves specifically uracil-containing ssDNA and dsDNA at 65 o C. The enzyme displays an optimal cleavage activity at 55-75 o C. Tba UDG cleaves DNA over a wide pH spectrum ranging from 4.0 to 9.0 with an optimal pH of 5.0-8.0. In addition, the Tba UDG activity is independent on a divalent metal ion; however, both Zn 2+ and Cu 2+ completely inhibits the enzyme activity. Furthermore, the Tba UDG activity is also inhibited by high NaCl concentration. Tba UDG removes uracil from DNA by the order: U≈U/G>U/T≈U/C>U/A. The mutational studies showed that both the E118A and N159A mutants completely abolish the cleavage activity and retain the compromised binding activity, suggesting that residues E118 and N159 in Tba UDG are important for uracil recognition and removal.Our work provides a basis for determining the role of Tba UDG in the base excision repair pathway for uracil repair in Thermococcus.

Endonuclease V (Endo V) is an important enzyme for repairing deoxyinosine in DNA. While bacterial and eukaryotic endo Vs have been well studied, knowledge of archaeal endo Vs is limited. Here, we first presented biochemical characterization of a thermostable endonuclease V from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba endo V). The recombinant enzyme possessed optimal endonuclease activity for cleaving deoxyinosine-containing DNA at 70-90 °C. Furthermore, Tba endo V can withstand 100 °C for 120 min without significant loss of its activity, suggesting the enzyme is thermostable. Tba endo V exhibited varying cleavage efficiencies at various pH levels from 6.0 to 11.0, among which an optimal pH for the enzyme was 8.0-9.0. In addition, a divalent metal ion was required for the enzyme to cleave DNA. Mn and Mg were optimal ions for the enzyme's activity whereas Ca, Zn and Co inhibited the enzyme activity. Moreover, the enzyme activity was suppressed by high NaCl concentration. Tba endo V bound to all DNA substrates; however, the enzyme exhibited a higher affinity for binding to deoxyinosine-containing DNA than normal DNA. Our work provides valuable information for revealing the role of Tba endo V in the base excision repair pathway for deoxyinosine repair in Thermococcus.

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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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.

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65~70 o C, and retains its DNA ligation activity even after heated at 100 o C for 2 hours, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0-10.0, and its optimal pH is 6.0-9.0. Tba ligase activity is dependent on a divalent metal ion: Mn 2+ , Mg 2+ or Ca 2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Remarkably, Tba ligase activity is independent on an additional nucleotide cofactor. However, ATP is required for Tba ligase activity when lowering the enzyme concentration. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylation, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. These observations suggest that the enzyme is an ATP-dependent DNA ligase. In addition, the mismatches at the first position 3′ to the nick suppress Tba ligase activity more than those at the first position 5′ to the nick. The enzyme also discriminates more effectively mismatches at 3′ to the nick than those at 5′ to the nick in ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.