Nucleic Acids Research, Vol 26, Issue 22 5157-5162, Copyright © 1998 by Oxford University Press
C Buhler, D Gadelle, P Forterre, JC Wang and A Bergerat
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.
ARTICLES
Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli
Institut de Genetique et Microbiologie, Batiment 409, Universite Paris Sud, CNRS UMR 2225, 91405 Orsay Cedex, France.
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