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.
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