Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli.
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.
Buhler
C
C
Institut de Génétique et Microbiologie, Batiment 409, Université Paris Sud, CNRS UMR 2225, 91405 Orsay Cedex, France.
Gadelle
D
D
Forterre
P
P
Wang
J C
JC
Bergerat
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006868:Hydrolysis; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics*
D019843:Archaeal Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C105141:DNA topoisomerase VI; D004250:DNA Topoisomerases, Type II