Intrinsic ATPase Activity of the B Subunit of DNA Topoisomerase VI from Sulfolobus Shibatae C. Buhler, P. Forterre, A Bergerat

Poster displayed during "The Eighth Conference on DNA Topoisomerases in Therapy" meeting held in Amsterdam,The Netherlands, October 15-17 1997.
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Intrinsic ATPase Activity of the B Subunit of DNA Topoisomerase VI from Sulfolobus Shibatae C. Buhler, P. Forterre and A Bergerat Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS URA 1354, 91405 Orsay Cedex, France. Recently we have found in the hyperthermophilic archaeon Sulfolobus shibatae a type II topoisomerase wich belongs to a new type II family of DNA topoisomerase. This enzyme called DNA topoisomerase VI is ATP dependant and is active in an heterotetramer form A2B2. No sequence similarities with the classical type II DNA topoisomerase family were found exept three small motifs (B1, B2 and B3) on the N-terminal sequence of the B subunit. These motives are not specific of type II DNA topoisomerases since they are also present in the proteins of the Hsp90 and MutL families. It has been previously shown that residues in the B1, B2, and B3 motifs on the E.coli DNA Gyrase B subunit are involved in the binding and hydrolysis of ATP. To test the implication of these motifs for ATP hydrolysis by type II-B DNA topoisomerase, we have overexpressed the B subunit of DNA topoisomerase VI from S. shibatae in E. coli strain BL21 lysS using the pet3 expression system. We have purified this recombinant B subunit to near homogeneity by heat treatment at 80°C of the total soluble proteins fraction and a phenyl sepharose chromatography. Using the pyruvate kinase / lactate dehydrogenase linked assay we have observed an intinsic ATPase activity by this recombinant B subunit at 80 °C. We are now performing site directed mutageneis on motifs B1 to B3 to identify residues implicated for ATP binding and hydrolysis.

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• Title
  
• Introduction 1
  
• Introduction 2
  
• Fig1: Conserved residues between type II DNA topoisomerases ans Hsp90 protein family and MutL protein family
  
• Fig2: Alignment around the uuuuExxxA motif in the C-terminal region of Topo II B-subunits
  
• Fig3: Overexpression of the S. shibatae DNA topoisomerase VI B-subunit in Escherichia coli .
  
• Fig4: Purification of the recombinant B subunit.
  
• Fig5: Preliminary results suggest that the B-subunit of DNA topoisomerase VI possesses an intrinsic ATPase activity at 75°C
  
• Fig6: Model for the evolution of type II DNA Topoisomerases.
  
• Conclusion/Perspectives.
  
• References.