Functional evaluation of four putative DNA-binding regions in Thermoanaerobacter tengcongensis reverse gyrase
- 147 Downloads
Reverse gyrase (RG) is an ATP-dependent type I DNA topoisomerase that introduces positive supercoils into DNA in thermophiles. Four regions of RG, i.e., the N-terminal zinc-finger motif, the β-hairpin in subdomain H1, the “latch”, and the C-terminal zinc-finger motif, were predicted to be involved in DNA binding previously. In this paper, the functions of these regions in the enzymatic activity were evaluated by mutational analysis of the Thermoanaerobacter tengcongensis reverse gyrase (TtRG). We demonstrated that TtRG exhibited positive-supercoiling activity only at high temperature (>50°C) and low salt concentration (~30 mM NaCl), and three of these four regions (except for the “latch”) were involved in DNA binding. Notably, mutations in the “latch” and β-hairpin regions of TtRG strongly impaired the ATPase activity, while mutations in the two zinc-finger motifs dramatically affected its thermal stability besides significant impairment of the DNA-binding ability. Accordingly, all of these four regions were found to be indispensable for the positive-supercoiling activity of TtRG. Taken together, we revealed that these putative DNA-contact regions affect the enzymatic activity of RG in different ways, and provided new insights into the structure and function of RG.
KeywordsReverse gyrase DNA binding Thermal stability ATPase activity Thermoanaerobacter tengcongensis
This work was supported by grants from the National Natural Science Foundation of China (NSFC) (Nos. 30621005, 30925001).
- Bouthier de la Tour C, Portemer C, Huber R, Forterre P, Duguet M (1991) Reverse gyrase in thermophilic eubacteria. J Bacteriol 173:3921–3923Google Scholar
- Bouthier de la Tour C, Amrani L, Cossard R, Neuman KC, Serre MC, Duguet M (2008) Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase. J Biol Chem 283:27395–27402Google Scholar
- Brown PO, Cozzarelli NR (1979) A sign inversion mechanism for enzymatic supercoiling of DNA. Science 206:1081–1083Google Scholar
- Hsieh TS, Plank JL (2009) Helicase-appended topoisomerases: new insight into the mechanism of directional strand-transfer. J Biol Chem 284(45):30737–30741Google Scholar
- Liu LF, Liu CC, Alberts BM (1980) Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break. Cell 19:697–707Google Scholar
- Wang JC (1996) DNA topoisomerases. Annu Rev Biochem 65:635–692Google Scholar