A key step in the DNA transport by type II DNA topoisomerase is the formation of a double-strand break with the enzyme being covalently linked to the broken DNA ends (referred to as the cleavage complex). In the present study, we have analyzed the formation and structure of the cleavage complex catalyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification procedure of a fully soluble recombinant topoVI was developed by expressing both subunits simultaneously in Escherichia coli. Using this recombinant enzyme, we observed that the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or AMP-PNP. This result suggests that ATP binding is required to stabilize an enzyme conformation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Similarly to other type II DNA topoisomerases, topoVI is covalently attached to the 5-ends of the broken DNA. However, sequence analysis of the doublestrand breaks revealed that they are all characterized by staggered two-nucleotide long 5 overhangs, contrasting with the four-base staggered double-strand breaks catalyzed by type IIA DNA topoisomerases. While no clear consensus sequences surrounding the cleavage sites could be described, interestingly A and T nucleotides are highly represented on the 5 extensions, giving a first insight on the preferred sequences recognized by this type II DNA topoisomerase.Type II DNA topoisomerases are ubiquitous enzymes that catalyze the ATP-dependent transport of one DNA duplex through a second DNA segment via a transient double-strand break (1). This ability to modulate the topological state of DNA is essential in major biological processes such as replication, recombination, and transcription (2). Until recently, these enzymes were thought to form a single family of homologous proteins. The discovery of DNA topoisomerase VI (topoVI) 1 in hyperthermophilic archaea has modified this classification. Type II DNA topoisomerases are now subdivided into two subfamilies, type IIA and IIB DNA topoisomerases (Fig. 1) (3).The type IIA subfamily contains three cellular representatives: eucaryotic DNA topoisomerase II (topoII), bacterial DNA gyrase, and DNA topoisomerase IV (topoIV). DNA gyrase and topoIV are heterotetramers composed of two subunits GyrA and GyrB, and ParC and ParE, respectively, while the eucaryotic enzyme is a homodimer. Despite this difference in quaternary structure, protein sequences comparison revealed that GyrB and ParE subunits are homologous to the N-terminal part of the eucaryotic enzyme while GyrA and ParC are homologous to the C-terminal half. These similarities were further confirmed by structural analysis of several fragments of Saccharomyces cerevisiae DNA topoII and Escherichia coli DNA gyrase (4 -7).Archaeal topoVI is the prototype of the recently described type IIB DNA topoisomerase subfamily (3). Thi...
Type II DNA topoisomerases have been classified into two families, Topo IIA and Topo IIB, based on structural and mechanistic dissimilarities. Topo IIA is the target of many important antibiotics and antitumoural drugs, most of them being inactive on Topo IIB. The effects and mode of action of Topo IIA inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. In contrast, studies of Topo IIB inhibitors were lacking. To document this field, we have studied two Hsp90 inhibitors (radicicol and geldanamycin), known to interact with the ATP-binding site of Hsp90 (the Bergerat fold), which is also present in Topo IIB. Here, we report that radicicol inhibits the decatenation and relaxation activities of Sulfolobus shibatae DNA topoisomerase VI (a Topo IIB) while geldanamycin does not. In addition, radicicol has no effect on the Topo IIA Escherichia coli DNA gyrase. In agreement with their different effects on DNA topoisomerase VI, we found that radicicol can theoretically fit in the ATP-binding pocket of the DNA topoisomerase VI ‘Bergerat fold’, whereas geldanamycin cannot. Radicicol inhibited growths of Sulfolobus acidocaldarius (a crenarchaeon) and of Haloferax volcanii (a euryarchaeon) at the same doses that inhibited DNA topoisomerase VI in vitro. In contrast, the bacteria E.coli was resistant to this drug. Radicicol thus appears to be a very promising compound to study the mechanism of Topo IIB in vitro, as well as the biological roles of these enzymes in vivo.
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