Functional evaluation of four putative DNA-binding regions in Thermoanaerobacter tengcongensis reverse gyrase

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.