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.
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