期刊
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
卷 134, 期 -, 页码 846-855出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijbiomac.2019.05.073
关键词
Thermococcus barophilus; Uracil DNA glycosylase; Base excision repair
资金
- Academic Leader of Middle and Young People of Yangzhou University Grant
- Open Project of State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University [MMLKFI8-05]
- practice innovation training program for college students in Yangzhou University [XKYCX18_072]
- Open Project of Key Laboratory of Marine Medicine, Guangdong Province
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences [2018011008]
Uracil DNA glycosylases (UDGs) play an important role in removing uracil from DNA to initiate DNA base excision repair. Here, we 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 exclusively uracil-containing ssDNA and dsDNA at 65 degrees C. The enzyme displays an optimal cleavage activity at 70-75 degrees C. Tba UDG cleaves DNA over a wide pH spectrum ranging from 4.0 to 11.0 with an optimal pH of 7.0-9.0. In addition, Tba UDG activity is independent on a divalent metal ion; however, both Zn2+ and Cu2+ completely inhibit the enzyme activity. Tba UDG activity is also inhibited by high NaCl concentration. Tba UDG removes uracil from DNA with the following preference: U approximate to U/G > U/T approximate to U/C > U/A. Kinetic results showed that Tba UDG cleaves uracil-containing ssDNA and dsDNA at a similar rate. The mutational studies showed that the E118A, N159A and H216A mutants completely abolish cleavage activity and retain compromised binding activity while the Y127A mutant displays similar cleavage and binding activities with the wild-type protein, suggesting that residues E118, N159 and H216 are essential for uracil removal and necessary for uracil recognition. (C) 2019 Published by Elsevier B.V.
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