, Volume 15, Issue 2, pp 281–291 | Cite as

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

Original Paper


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.


Reverse 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).


  1. Atomi H, Matsumi R, Imanaka T (2004) Reverse gyrase is not a prerequisite for hyperthermophilic life. J Bacteriol 186:4829–4833PubMedCrossRefGoogle Scholar
  2. Bao Q et al (2002) A complete sequence of the T. tengcongensis genome. Genome Res 12:689–700PubMedCrossRefGoogle Scholar
  3. 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
  4. 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
  5. Brochier-Armanet C, Forterre P (2007) Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers. Archaea 2:83–93PubMedCrossRefGoogle Scholar
  6. Brown PO, Cozzarelli NR (1979) A sign inversion mechanism for enzymatic supercoiling of DNA. Science 206:1081–1083Google Scholar
  7. Campbell BJ et al (2009) Adaptations to submarine hydrothermal environments exemplified by the genome of Nautilia profundicola. PLoS Genet 5:e1000362PubMedCrossRefGoogle Scholar
  8. Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369–413PubMedCrossRefGoogle Scholar
  9. Dekker NH et al (2002) The mechanism of type IA topoisomerases. Proc Natl Acad Sci USA 99:12126–12131PubMedCrossRefGoogle Scholar
  10. Forterre P (2002) A hot story from comparative genomics: reverse gyrase is the only hyperthermophile-specific protein. Trends Genet 18:236–237PubMedCrossRefGoogle Scholar
  11. Heine M, Chandra SB (2009) The linkage between reverse gyrase and hyperthermophiles: a review of their invariable association. J Microbiol 47:229–234PubMedCrossRefGoogle Scholar
  12. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68PubMedCrossRefGoogle Scholar
  13. Hsieh TS, Capp C (2005) Nucleotide- and stoichiometry-dependent DNA supercoiling by reverse gyrase. J Biol Chem 280:20467–20475PubMedCrossRefGoogle Scholar
  14. Hsieh TS, Plank JL (2006) Reverse gyrase functions as a DNA renaturase: annealing of complementary single-stranded circles and positive supercoiling of a bubble substrate. J Biol Chem 281:5640–5647PubMedCrossRefGoogle Scholar
  15. 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
  16. Jungblut SP, Klostermeier D (2007) Adenosine 5′-O-(3-thio)triphosphate (ATPgammaS) promotes positive supercoiling of DNA by T. maritima reverse gyrase. J Mol Biol 371:197–209PubMedCrossRefGoogle Scholar
  17. Kampmann M, Stock D (2004) Reverse gyrase has heat-protective DNA chaperone activity independent of supercoiling. Nucleic Acids Res 32:3537–3545PubMedCrossRefGoogle Scholar
  18. Kikuchi A, Asai K (1984) Reverse gyrase—a topoisomerase which introduces positive superhelical turns into DNA. Nature 309:677–681PubMedCrossRefGoogle Scholar
  19. Li S, Wilkinson MF (1997) Site-directed mutagenesis: a two-step method using PCR and DpnI. Biotechniques 23:588–590PubMedGoogle Scholar
  20. 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
  21. Nakasu S, Kikuchi A (1985) Reverse gyrase; ATP-dependent type I topoisomerase from Sulfolobus. EMBO J 4:2705–2710PubMedGoogle Scholar
  22. Napoli A et al (2004) Reverse gyrase recruitment to DNA after UV light irradiation in Sulfolobus solfataricus. J Biol Chem 279:33192–33198PubMedCrossRefGoogle Scholar
  23. Perugino G, Valenti A, D’Amaro A, Rossi M, Ciaramella M (2009) Reverse gyrase and genome stability in hyperthermophilic organisms. Biochem Soc Trans 37:69–73PubMedCrossRefGoogle Scholar
  24. Rodriguez AC (2002) Studies of a positive supercoiling machine. Nucleotide hydrolysis and a multifunctional “latch” in the mechanism of reverse gyrase. J Biol Chem 277:29865–29873PubMedCrossRefGoogle Scholar
  25. Rodriguez AC (2003) Investigating the role of the latch in the positive supercoiling mechanism of reverse gyrase. Biochemistry 42:5993–6004PubMedCrossRefGoogle Scholar
  26. Rodriguez AC, Stock D (2002) Crystal structure of reverse gyrase: insights into the positive supercoiling of DNA. EMBO J 21:418–426PubMedCrossRefGoogle Scholar
  27. Sakai-Kato K, Umezawa Y, Mikoshiba K, Aruga J, Utsunomiya-Tate N (2009) Stability of folding structure of Zic zinc finger proteins. Biochem Biophys Res Commun 384:362–365PubMedCrossRefGoogle Scholar
  28. Schoeffler AJ, Berger JM (2008) DNA topoisomerases: harnessing and constraining energy to govern chromosome topology. Q Rev Biophys 41:41–101PubMedCrossRefGoogle Scholar
  29. Shibata T, Nakasu S, Yasui K, Kikuchi A (1987) Intrinsic DNA-dependent ATPase activity of reverse gyrase. J Biol Chem 262:10419–10421PubMedGoogle Scholar
  30. Slesarev AI, Kozyavkin SA (1990) DNA substrate specificity of reverse gyrase from extremely thermophilic archaebacteria. J Biomol Struct Dyn 7:935–942PubMedGoogle Scholar
  31. Valenti A, Napoli A, Ferrara MC, Nadal M, Rossi M, Ciaramella M (2006) Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus. Nucleic Acids Res 34:2098–2108PubMedCrossRefGoogle Scholar
  32. Valenti A et al (2008) Dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine. Nucleic Acids Res 36:4587–4597PubMedCrossRefGoogle Scholar
  33. Wang JC (1996) DNA topoisomerases. Annu Rev Biochem 65:635–692Google Scholar
  34. Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3:430–440PubMedCrossRefGoogle Scholar
  35. Xue Y, Xu Y, Liu Y, Ma Y, Zhou P (2001) Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int J Syst Evol Microbiol 51:1335–1341PubMedGoogle Scholar

Copyright information

© Springer 2011

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China

Personalised recommendations