caa a22 4500 445818778 CHVBK 20180317145237.0 cr unu---uuuuu 170323e20110301xx s 000 0 eng 10.1007/s00792-011-0356-5 doi (NATIONALLICENCE)springer-10.1007/s00792-011-0356-5 Functional evaluation of four putative DNA-binding regions in Thermoanaerobacter tengcongensis reverse gyrase [Elektronische Daten] [Jie Li, Jingfang Liu, Jian Zhou, Hua Xiang] 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 (~30mM 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. Springer, 2011 Reverse gyrase nationallicence DNA binding nationallicence Thermal stability nationallicence ATPase activity nationallicence Thermoanaerobacter tengcongensis nationallicence Li Jie State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Liu Jingfang State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Zhou Jian State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Xiang Hua State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Extremophiles Springer Japan 15/2(2011-03-01), 281-291 1431-0651 15:2<281 2011 15 792 https://doi.org/10.1007/s00792-011-0356-5 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00792-011-0356-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Li Jie State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 700 1- Liu Jingfang State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 700 1- Zhou Jian State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 700 1- Xiang Hua State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 773 0- Extremophiles Springer Japan 15/2(2011-03-01), 281-291 1431-0651 15:2<281 2011 15 792 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer