DNA Topoisomerase VI Generates ATP-dependent Double-strand Breaks with Two-nucleotide Overhangs

Abstract

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...

Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol

“…For in vitro tests, the drugs were diluted in DMSO. S.shibatae DNA topoisomerase VI was purified as a heterotetramer after co-expression and overproduction of the two subunits, Top6A and Top6B in Escherichia coli , as previously described (11). E.coli DNA gyrase was purchased from TopoGEN.…”

“…In contrast, the nicking-closing modules of Topo IIA and Topo IIB are non-homologous, being structurally dissimilar (10). Furthermore, the production of DSB by Topo IIA is ATP independent and generates single-stranded extension of 4 bp, whereas DSB formation by Topo IIB is ATP-dependent and generates single-stranded extension of 2 bp (11). These data indicate that the type II DNA topoisomerase activity has been invented twice in the course of evolution by the independent recruitment of two different nicking-closing modules to work with the same type of ATP-binding module (4).…”

Functional characterization of the meiosis-specific DNA double-strand break inducing factor SPO-11 from C. elegans

“…We addressed this question by incubating SPO-11 with negatively supercoiled DNA under a variety of reaction conditions, followed by deproteinization of the reaction mixtures and analysis in an agarose gel 28 . We did not observe any DNA cleavage product regardless of whether Mg 2+ , ATP, or Mg 2+ -ATP was added or not (Fig.…”

A genetic screen for mutants defective in IAA1-LUC degradation inArabidopsis thalianareveals an important requirement forTOPOISOMERASE6Bin auxin physiology

“…24 The A subunits catalyze the double-stranded DNA cleavage reaction. 25,26 The B subunits contain a GHKL-type (gyrase, Hsp90, histidine kinase, MutL) ATPase domain, 24,27 a transducer domain, a helixturn-helix domain, and a C-terminal domain. 26,28 The transducer domain links the B subunit to the A subunit and also contains a critical lysyl residue that contacts the g-phosphate of ATP bound in the GHKL-domain.…”