Elsevier

Biochimie

Volume 89, Issue 4, April 2007, Pages 447-455
Biochimie

Reverse gyrase: An insight into the role of DNA-topoisomerases

https://doi.org/10.1016/j.biochi.2006.12.010Get rights and content

Abstract

Reverse gyrase was discovered more than twenty years ago. Recent biochemical and structural results have greatly enhanced our understanding of their positive supercoiling mechanism. In addition to new biochemical properties, a fine tuning of reverse gyrase regulation in response to DNA damaging agents has been recently described. These data give us a new insight in the cellular role of reverse gyrase. Moreover, it has been proposed that reverse gyrase has been implicated in genome stability.

Introduction

If the base pairing of the two DNA strands proposed by Watson and Crick in 1953 [1], [2] quickly appeared as an amazing interaction sufficient to explain the flow of genetic information in the cell, the double helical structure of DNA brought serious problems as mentioned by Watson and Crick themselves: “Since the two chains in our model are intertwined, it is essential for them to untwist if they are to separate. … Although it is difficult at the moment to see how these processes occur without everything getting tangled, we do not feel that this objection will be insuperable.” [2]. This problem gave a new scientific field, the DNA topology, and allowed the studies of fascinating enzymes, the DNA topoisomerases. The first DNA topoisomerase, named ω protein, was discovered in Escherichia coli by Jim Wang in 1971 and is now called topoisomerase I [3]. One year later, James Champoux and Robert Dulbecco discovered a nicking-closing activity from nuclear extracts of mouse cells which was also named topoisomerase I [4]. However, these topoisomerases are quite different since “nicking-closing” enzyme could relax both negatively and positively supercoiled DNA while ω protein could relax only negatively supercoiled DNA. Now, these enzymes are classified respectively as type IB and type IA. Few years later, Martin Gellert and co-workers discovered DNA gyrase, from Escherichia coli [5]. This topoisomerase required the presence of ATP and is able to negatively supercoil DNA. Homologous topoisomerases which hydrolyze ATP but cannot supercoil DNA were discovered from eukarya at the end of the seventies and were named topoisomerase II. Then, very quickly, the number of these enzymes increased and the importance of their key role in all the transactions of genetic information was highlighted [6], [7], [8]. In association with other proteins that bind DNA like histones, SSB, helicases etc., the work of the topoisomerases is to shape DNA [9]. To change the topology of DNA, topoisomerases, called “magicians” by Jim Wang, performed a transient breaking and rejoining of DNA strands that correspond to two transesterification reactions. After the first transesterification reaction the topoisomerase is covalently linked to DNA by a phosphotyrosyl bond [10]. Type I topoisomerases transiently cleave one DNA strand which allows another single strand to pass through this breakage. Type II topoisomerases bind on two different DNA segments. They transiently cleave the two DNA strands of the “G” segment which allows another duplex, the transport “T” segment, to be transported through the gate (G segment). These strands passage reactions are followed by a second transesterification that reseal the DNA break.

Both type I and type II topoisomerases are present in all living cells and obviously exist since Nature invented DNA. However, comparison of the DNA-topoisomerases issued from Bacteria and Eukarya indicated that these enzymes are quite different. In Bacteria, a homeostatic control of DNA supercoiling has been proposed [11]. This is realized by two antagonistic topoisomerase activities: the relaxation of negatively supercoiled DNA is catalyzed by ω protein and the negative supercoiling of relaxed DNA is catalyzed by gyrase. In Eukarya, both type I and type II enzymes catalyze the relaxation of negatively supercoiled as well as positively supercoiled DNA. However, in Eukarya, naked DNA appears negatively supercoiled but this torsion is constrained by nucleosomes. Though the topological problems of DNA in Bacteria and Eukarya seem very similar, why have these organisms developed two different strategies to solve topological constraints? Another formulation would be: What is essential for DNA transactions pathways and consequently, what could be similar in all living cells? What is the result of adaptation processes?

To answer these questions, Michel Duguet and Gilles Mirambeau in collaboration with Patrick Forterre in France and Akihiko Kikuchi and Keiko Asai in Japan independently initiated the characterization of DNA topoisomerases issued from the third domain of life, the Archaea. At this juncture, the concept of Archaea had just emerged [12] and these researchers took advantage of the biodiversity of living cells to get a better understanding of DNA topoisomerases. In this context, hyperthermophilic sulfur dependant Archaea discovered some years sooner by Thomas Brock [13] were the most exciting because they live in demoniac conditions: above 80 °C and at a pH range from 1 to 4! Of course, at this temperature, DNA must face strands melting and consequently need to adapt its DNA topology to this temperature.

In 1984 while Mirambeau et al. described the properties of the major topoisomerase activity present in sulfolobales [14], an ATP-and magnesium dependant relaxation activity, Kikuchi and Asai described a new topoisomerase activity able to positively supercoil DNA in the presence of ATP-and magnesium and called this new and amazing DNA topoisomerase reverse gyrase [15]. The relevance of this positive supercoiling activity was demonstrated two years later by showing that highly positive supercoiled DNA exist in a hyperthermophilic virus, the Sulfolobus shibatae Virus 1 (SSV1) [16]. Further studies of this topoisomerase gave a lot of unexpected results and certainly contributed to modify the view of DNA topology. Indeed, positive supercoiling exists in all organisms. It is generated as a consequence of tracking processes of dynamic DNA machineries that move along DNA axes such as replication or transcription machineries. Topoisomerases must efficiently eliminate these supercoils to prevent arrest of these machineries. Finally, reverse gyrase rapidly appeared as a hallmark of all hyperthermophilic organisms—indeed, by using antibodies raised against reverse gyrase, we found it in all hyperthermophilic Archaea tested [17]. Moreover reverse gyrase activity is also found in all hyperthermophilic organisms, in Archaea as well as in Bacteria [18], [19]. More recently, genome sequencing showed that genes encoding reverse gyrase seem to be present in all thermophilic organisms but such genes were never found in mesophiles or psychrophiles. Consequently, this gene could be considered as the marker of life at high temperature [20]. Reverse gyrase is not only conserved in hyperthermophilic Archaea but also in hyperthermophilic Eubacteria [21], suggesting a lateral gene transfer [22].

Section snippets

Purification of reverse gyrase

Reverse gyrase was first discovered in Sulfolobus species and is the most abundant DNA topoisomerase in this cell [14], [15]. Purification of this enzyme was performed from different hyperthermophilic archaeal strains including crenarcheota [23], [24], [25], [26] and euryarcheota [27], [28]. The reverse gyrase activity was also detected in all hyperthermophilic Eubacteria tested [19] and the enzymes purified from Calderobacterium hydrogenophilum or Thermotoga maritima share the same

Reverse gyrase assay

In vitro, reverse gyrase catalyzes both the relaxation of negatively supercoiled DNA and the positive supercoiling of DNA. These two reactions correspond to an increasing of the linking number of DNA, differing only by the extent of the reaction. Reverse gyrase performs a positive gyration per se because it introduces additional positive superturns in a relaxed or slightly positive supercoiled DNA substrate [24], [34]. The existence of positively supercoiled DNA was demonstrated by

Role of the nucleotide

Reverse gyrase requires a very low amount of ATP since 10 μM is sufficient to relax negatively supercoiled DNA or to positively supercoil relaxed DNA [34]. In the course of these reactions, ATP is hydrolyzed into ADP, in a DNA dependent manner [39]. ATP hydrolysis is more efficient in the presence of single-stranded DNA than in the presence of double-stranded DNA [39]. dATP can be used instead of ATP with the same efficiency [39], [40]. DNA relaxation and production of positive supercoils is

DNA binding

By using covalent closure of nicked DNA by thermophilic ligase it has been shown that binding of reverse gyrase to DNA induces an increasing of the helical repeat of DNA [29]. This DNA unwinding is observed in the absence of ATP and can be used to titrate the enzyme bound efficiently to DNA. The unwinding effect is also recovered by using a mutant inactivated by the replacement of the tyrosine at the active site by a phenylalanine. This indicates that unwinding is not induced by the

Primary structure

Important advance on reverse gyrase understanding is due to its gene sequencing. Using antibodies raised against Sulfolobus acidocaldarius reverse gyrase [24], we have cloned and then sequenced the first reverse gyrase gene [31]. The deduced amino-acid sequence clearly showed that this polypeptide is a chimera between a classical type IA topoisomerase corresponding to the C-terminal part and an ATPase related to the helicase superfamily 2 (SF2) corresponding to the N-terminal part (see Fig. 1).

Three-dimensional structure

The determination of the three-dimensional structure of full-length reverse gyrase from Archeoglobus fulgidus [54] shows that it has a padlock-like shape with the same four parts forming the ring typical of type IA topoisomerase fold [55], [56], [57]. M. Duguet had recently proposed that this ring structure results from an inverted repetition of two topofolds where only one of them contains the catalytic tyrosine residue and the second one contains arginine residues facing the first. These

Mechanism of reverse gyration

Both the primary structure and three-dimensional structure of reverse gyrase clearly indicate that it is a combination of a type IA topoisomerase and a SF2 protein. In addition, biochemical data prompt us to propose the two domains model [32]. Briefly, in a covalently closed circular molecule, the DNA is unwound by the SF2 domain giving partially single-stranded DNA tightly bound to the protein. The unwinding induces a positive superturn in the rest of the molecule while unwound DNA is relaxed

Role and regulation of reverse gyrase

At least one reverse gyrase encoding gene is found in all hyperthermophilic organisms, suggesting a very important role in these organisms. In Thermococcus kodakaraensis, the inactivation of its gene is not lethal but meanwhile, its importance appears more and more when the growth temperature increased. At the optimal growth temperature, the inactivation of reverse gyrase gene considerably increased the doubling time compared with wild type [64]. This important result indicates a particularly

Conclusion

Since 1984, the year of the discovery of reverse gyrase, a lot of results improved our knowledge of this fascinating DNA topoisomerase. Important efforts were made to enhance the understanding of the positive supercoiling mechanism. Further experiments are necessary to understand the precise role of reverse gyrase in thermophilic organisms. However, positive supercoiling activity could exist in mesophilic organisms since it was reported in human cells [83] and more recently as a property of an

Acknowledgments

I thank Christiane Elie, Sharon Larmony and Samia Salhi for critical reading of the manuscript. The Microbiology team of the Laboratoire de Génétique et de Biologie Cellulaire is supported by funds from Université Versailles-St-Quentin-en-Yvelines, Centre National de la Recherche Scientifique (CNRS) and EDF (Comité de radioprotection).

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