Archaea: A Gold Mine for Topoisomerase Diversity.

The control of DNA topology is a prerequisite for all the DNA transactions such as DNA replication, repair, recombination, and transcription. This global control is carried out by essential enzymes, named DNA-topoisomerases, that are mandatory for the genome stability. Since many decades, the Archaea provide a significant panel of new types of topoisomerases such as the reverse gyrase, the type IIB or the type IC. These more or less recent discoveries largely contributed to change the understanding of the role of the DNA topoisomerases in all the living world. Despite their very different life styles, Archaea share a quasi-homogeneous set of DNA-topoisomerases, except thermophilic organisms that possess at least one reverse gyrase that is considered a marker of the thermophily. Here, we discuss the effect of the life style of Archaea on DNA structure and topology and then we review the content of these essential enzymes within all the archaeal diversity based on complete sequenced genomes available. Finally, we discuss their roles, in particular in the processes involved in both the archaeal adaptation and the preservation of the genome stability.

Direct observation of helicase-topoisomerase coupling within reverse gyrase.

Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.

The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus.

Maintaining an appropriate DNA topology with DNA-based processes (DNA replication, transcription and recombination) is crucial for all three domains of life. In bacteria, the homeostatic regulation for controlling DNA supercoiling relies on antagonistic activities of two DNA topoisomerases, TopoI and gyrase. In hyperthermophilic crenarchaea, the presence of such a regulatory system is suggested as two DNA topoisomerases, TopoVI and reverse gyrase, catalyze antagonistic activities. To test this hypothesis, we estimated and compared the number of the TopoVI with that of the two reverse gyrases, TopR1 and TopR2, in Sulfolobus solfataricus cells maintained either at 80 or at 88°C, or reciprocally shifted from one temperature to the other. From the three DNA topoisomerases, TopR1 is the only one exhibiting significant quantitative variations in response to the up- and down-shifts. In addition, the corresponding intrinsic activities of these three DNA topoisomerases were tested in vitro at both temperatures. Although temperature modulates the three DNA topoisomerases activities, TopR1 is the sole topoisomerase able to function at high temperature. Altogether, results presented in this study demonstrate, for the first time, that the DNA topological state of a crenarchaeon is regulated via a homeostatic control, which is mainly mediated by the fine-tuning of TopR1.

Type IA DNA Topoisomerases: A Universal Core and Multiple Activities.

All the type IA topoisomerases display universal characteristics relying on a core region basically responsible for the transesterification and the strand passage reaction. First limited to the bacterial domain for a long time, these enzymes were further retrieved in Archaea and Eukarya as well. This is representative of an extremely ancient origin, probably due to an inheritance from the RNA world. As remaining evidence, some current topoisomerases IA have retained a RNA topoisomerase activity. Despite the presence of this core region in all of these TopoIAs, some differences exist and are originated from variable regions, located essentially within both extremities, conferring on them their specificities. During the last 2 decades the evidence of multiple activities and dedicated roles highlighted the importance of the topoisomerases IA. It is now obvious that topoisomerases IA are key enzymes involved in the maintenance of the genome stability. The discovery of these new activities was done thanks to the use of more accurate assays, based on new sophisticated DNA substrates.

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.

A specific proteomic response of Sulfolobus solfataricus P2 to gamma radiations.

Sulfolobus solfataricus is an acidophilic hyperthermophilic crenarchaeon living at 80 °C in aerobic conditions. As other thermophilic organisms, S. solfataricus is resistant to gamma irradiation and we studied the response of this microorganism to this ionizing irradiation by monitoring cell growth, DNA integrity and proteome variations. In aerobic conditions, the S. solfataricus genome was fragmented due to the multiple DNA double strand breakages induced by γ-rays and was fully restored within a couple of hours. Comparison of irradiated and unirradiated cell proteomes indicated that only few proteins changed. The proteins identified by mass spectrometry are involved in different cellular pathways including DNA replication, recombination and repair. Interestingly, we observed that some proteins are irradiation dose-specific while others are common to the cell response regardless of the irradiation dose. Most of the proteins highlighted in these conditions seem to act together to allow an efficient cell response to γ-irradiation.

Insight into the cellular involvement of the two reverse gyrases from the hyperthermophilic archaeon Sulfolobus solfataricus.

BACKGROUND: Reverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We showed previously that the two genes are differently regulated according to temperature and that the corresponding purified recombinant reverse gyrases have different enzymatic characteristics. These observations suggest a specialization of functions of the two reverse gyrases. As no mutants of the TopR genes could be obtained in Sulfolobales, we used immunodetection techniques to study the function(s) of these proteins in S. solfataricus in vivo. In particular, we investigated whether one or both reverse gyrases are required for the hyperthermophilic lifestyle. RESULTS: For the first time the two reverse gyrases of S. solfataricus have been discriminated at the protein level and their respective amounts have been determined in vivo. Actively dividing S. solfataricus cells contain only small amounts of both reverse gyrases, approximately 50 TopR1 and 125 TopR2 molecules per cell at 80°C. S. solfataricus cells are resistant at 45°C for several weeks, but there is neither cell division nor replication initiation; these processes are fully restored upon a return to 80°C. TopR1 is not found after three weeks at 45°C whereas the amount of TopR2 remains constant. Enzymatic assays in vitro indicate that TopR1 is not active at 45°C but that TopR2 exhibits highly positive DNA supercoiling activity at 45°C. CONCLUSIONS: The two reverse gyrases of S. solfataricus are differently regulated, in terms of protein abundance, in vivo at 80°C and 45°C. TopR2 is present both at high and low temperatures and is therefore presumably required whether cells are dividing or not. By contrast, TopR1 is present only at high temperature where the cell division occurs, suggesting that TopR1 is required for controlling DNA topology associated with cell division activity and/or life at high temperature. Our findings in vitro that TopR1 is able to positively supercoil DNA only at high temperature, and TopR2 is active at both temperatures are consistent with them having different functions within the cells.

TopR2, the second reverse gyrase of Sulfolobus solfataricus, exhibits unusual properties.

Whereas reverse gyrase is considered as a strong marker of thermophily, the function of this peculiar type IA topoisomerase still remains to be elucidated. The archaeon Sulfolobus solfataricus encodes two reverse gyrases, TopR1 and TopR2. This duplication seems to be important because most of Crenarcheota exhibit two copies of reverse gyrase. However, to date, while TopR1 has been well characterized, no characterization of TopR2 has been reported. In this study, we describe for the first time the activity of S. solfataricus TopR2 that appears as a new reverse gyrase. Indeed, in spite of the sequence similarities between TopR1 and TopR2, we evidence unexpected great differences between the two enzymes. While TopR1 exhibits ATP-independent relaxation activity, TopR2 does not, and its activity is strictly dependent on the presence of ATP. Whereas TopR1 is a distributive topoisomerase, TopR2 exhibits an amazing high intrinsic processivity compared to all the topoisomerases studied so far. TopR2 is able to introduce a very high number of positive superturns in DNA, while TopR1 generates weakly positively supercoiled DNA. Finally, TopR2 behaves differently from TopR1 when incubated at different assay temperatures. All the results presented in this study indicate that TopR1 and TopR2 have, in vitro, different activities suggesting different functions in vivo.

Transcriptional analysis of the two reverse gyrase encoding genes of Sulfolobus solfataricus P2 in relation to the growth phases and temperature conditions.

Sulfolobus solfataricus, a hyperthermophilic crenarchaeon, contains two genes encoding reverse gyrases, topR1 and topR2. The steady-state level of their transcripts were quantified during the growth phases for cells maintained either at 72, or 80 degrees C, and after temperature changes from one to the other temperature. The transcripts of both genes are weakly expressed, but the highest level is observed in actively dividing cells, and is almost undetectable in cells in decline phase. During the temperature shift experiments, there is no significant topR2 variation. By contrast, there is a maximum 2.4-fold increase in topR1 transcripts within 30 min after the downshift. After 1 h, the transcript level reaches the level characteristic of cells adapted to the new temperature. After an upward shift, the topR1 expression pattern is inversely regulated with a transient decrease with the same time course. The topR1 expression profile is completely different from that of topR2 after temperature shift experiments; this suggests a different regulation process for the two reverse gyrase genes. The fine tuning of the topR1 transcript expression within a short interval of time after a temperature shift illustrates a rapid adaptation response to temperature change.

Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus.

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.

Reverse gyrase recruitment to DNA after UV light irradiation in Sulfolobus solfataricus.

Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair.