Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli

Abstract

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.

Reverse Gyrase, the Two Domains Intimately Cooperate to Promote Positive Supercoiling

“…Reconstitution of topoisomerase activities from separate subunits has been obtained in several cases (13,25), but it is one of the first examples of reconstitution from separately expressed putative domains designed solely on the basis of amino acid sequence comparisons. This result indicates that the separate domains are able to naturally adopt a fold similar to their fold within the full-length reverse gyrase.…”

Identification, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia

“…This led to the classification of type II DNA topoisomerases into two evolutionary distinct protein families, type IIA and type IIB. Top VI belongs to type IIB family, others belong to type IIA [5].…”

The “GyrA-box” Is Required for the Ability of DNA Gyrase to Wrap DNA and Catalyze the Supercoiling Reaction

“…Type I topoisomerases alter the linking number in steps of one by breaking one strand of duplex DNA, passing the other strand through the break, and then resealing the broken strand, whereas type II topoisomerases alter the linking number in steps of two by breaking both strands, passing another segment of the helix through the break, and then resealing the broken strands. The discovery of a novel type II enzyme from Sulfolobus shibatae (3,4) prompted the division of the type II topoisomerases into the type IIA and type IIB subtypes. All type II topoisomerases, except the S. shibatae topoisomerase VI, belong to type IIA subtype.…”