The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.     

DNA supercoiling and transcription in Escherichia coli: The FIS connection

The nucleoid-associated protein FIS modulates the topology of DNA in a growth-phase dependent manner functioning homeostatically to counteract excessive levels of negative superhelicity. We propose that this is achieved by at least two mechanisms: the physical constraint of low levels of negative superhelicity by FIS binding to DNA and by a reduction in the expression and effectiveness of DNA gyrase. In addition, high levels of expression of the fis gene do themselves require a high negative superhelical density. On DNA substrates containing phased high affinity binding sites, as exemplified by the upstream activating sequence of the tyrT promoter, FIS forms tightly bent DNA structures, or microloops, that are necessary for the optimal expression of the promoter. We suggest that these microloops compensate in part for the FIS-induced lowering of the superhelical density.     

Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes

Escherichia coli deletion mutants lacking DNA topoisomerase I have been identified previously and shown to grow at a normal rate. We show that such strains grow normally only because of spontaneously arising mutations that compensate for the topoisomerase I defect. Several of these compensatory mutations have been found to map at or near the genes encoding DNA gyrase, gyrA and gyrB. DNA gyrase assays of crude extracts show that strains carrying the mutations have lower gyrase activity. Thus the mutations are in the gyrase structural genes or in nearby regulatory sequences. These results, in conjunction with DNA supercoiling measurements of others, indicate that in vivo DNA superhelicity is a result of a balance between topoisomerase I and gyrase activities. An excess of negative supercoils due to an absence of topoisomerase I is deleterious to the cell, but a moderate gyrase deficiency is not harmful.     

An architectural role of the Escherichia coli chromatin protein FIS in organising DNA

The Escherichia coli chromatin protein FIS modulates the topology of DNA in a growth phase-dependent manner. In this study we have investigated the global effect of FIS binding on DNA architecture in vitro. We show that in supercoiled DNA molecules FIS binds at multiple sites in a non-random fashion and increases DNA branching. This global DNA reshaping effect is independent of the helical phasing of FIS binding sites. We propose, in addition to the previously inferred stabilisation of tightly bent DNA microloops in the upstream regions of certain promoters, that FIS may perform the distinct architectural function of organising branched plectonemes in the E.coli nucleoid.     

DNA gyrase, topoisomerase IV, and the 4-quinolones.

For many years, DNA gyrase was thought to be responsible both for unlinking replicated daughter chromosomes and for controlling negative superhelical tension in bacterial DNA. However, in 1990 a homolog of gyrase, topoisomerase IV, that had a potent decatenating activity was discovered. It is now clear that topoisomerase IV, rather than gyrase, is responsible for decatenation of interlinked chromosomes. Moreover, topoisomerase IV is a target of the 4-quinolones, antibacterial agents that had previously been thought to target only gyrase. The key event in quinolone action is reversible trapping of gyrase-DNA and topoisomerase IV-DNA complexes. Complex formation with gyrase is followed by a rapid, reversible inhibition of DNA synthesis, cessation of growth, and induction of the SOS response. At higher drug concentrations, cell death occurs as double-strand DNA breaks are released from trapped gyrase and/or topoisomerase IV complexes. Repair of quinolone-induced DNA damage occurs largely via recombination pathways. In many gram-negative bacteria, resistance to moderate levels of quinolone arises from mutation of the gyrase A protein and resistance to high levels of quinolone arises from mutation of a second gyrase and/or topoisomerase IV site. For some gram-positive bacteria, the situation is reversed: primary resistance occurs through changes in topoisomerase IV while gyrase changes give additional resistance. Gyrase is also trapped on DNA by lethal gene products of certain large, low-copy-number plasmids. Thus, quinolone-topoisomerase biology is providing a model for understanding aspects of host-parasite interactions and providing ways to investigate manipulation of the bacterial chromosome by topoisomerases.     

Control of DNA topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus

Plasmid topology varies transiently in hyperthermophilic archaea during thermal stress. As in mesophilic bacteria, DNA linking number (Lk) increases during heat shock and decreases during cold shock. Despite this correspondence, plasmid DNA topology and proteins presumably involved in DNA topological control in each case are different. Plasmid DNA in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typical of bacteria, eukaryotes and mesophilic archaea. We have analysed the regulation of DNA topological changes during thermal stress in Sulfolobus islandicus (kingdom Crenarchaeota), which harbours two plasmids, pRN1 and pRN2. In parallel with plasmid topological variations, we analysed levels of reverse gyrase, topoisomerase VI (Topo VI) and the small DNA-binding protein Sis7, as well as topoisomerase activities in crude extracts during heat shock from 80 degrees C to 85-87 degrees C, and cold shock from 80 degrees C to 65 degrees C. Quantitative changes in reverse gyrase, Topo VI and Sis7 were not significant. In support of this, inhibition of protein synthesis in S. islandicus during shocks did not alter plasmid topological dynamics, suggesting that an increase in topoisomerase levels is not needed for control of DNA topology during thermal stress. A reverse gyrase activity was detected in crude extracts, which was strongly dependent on the assay temperature. It was inhibited at 65 degrees C, but was greatly enhanced at 85 degrees C. However, the intrinsic reverse gyrase activity did not vary with heat or cold shock. These results suggest that the control of DNA topology during stress in Sulfolobus relies primarily on the physical effect of temperature on topoisomerase activities and on the geometry of DNA itself. Additionally, we have detected an enhanced thermoresistance of reverse gyrase activities in cultures subject to prolonged heat shock (but not cold shock). This acquired thermotolerance at the enzymatic level is abolished when cultures are treated with puromycin, suggesting a requirement for protein synthesis.     

Control of bacterial DNA supercoiling

Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, supercoiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady-state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.     

Effect of DNA topology on plasmid DNA repair in vivo

Escherichia coli nucleotide excision repair (NER) is responsible for removing bulky DNA adducts by dual incisions of the UvrABC endonuclease. Although the activity of the UvrAB complex which can induce DNA conformational change is employed in NER, the involvement of DNA topology and DNA topoisomerases remains unclear. We examined the effect of topoisomerase inhibitions on a NER in vivo system. The repair analysis of intracellular plasmid revealed that the DNA damage on positive supercoils generated by gyrase inhibition remained unrepaired, whereas the DNA damage was repaired in topoisomerase I mutants. These results suggest that DNA topology affects the NER process and the removal of positive supercoils by gyrase is vital for the efficiency of the E. coli NER system.     

Isoleucine 10 is essential for DNA gyrase B function in Escherichia coli

DNA gyrase is an essential enzyme that regulates the DNA topology in bacteria. It belongs to the type II DNA topoisomerase family and is responsible for the introduction of negative supercoils into DNA at the expense of hydrolysis of ATP molecules. The aim of the present work was to study the contribution of I10, one of the most important residues responsible for the stabilization of GyrB dimer and involved in the ATP-binding step, in the ATP-hydrolysis reaction and in the DNA supercoiling mechanism. We constructed MBP-tagged GyrB mutants I10G and Delta4-14. Our results demonstrate that both mutations severely affect the DNA-dependent ATPase activity and DNA supercoiling. Mutation of Y5 residue involved in the formation of ATPase catalytic site (Y5G mutant) had only little effect on the DNA-dependent ATPase activity and DNA supercoiling. Interestingly, the DNA-relaxation activity of MBP-GyrB mutants and wild type was completely inhibited by ATP. Binding of ADPNP to MBP-tagged mutants was significantly decreased. ADPNP had no effect on DNA-relaxation activity of MBP-tagged mutants but was able to inhibit MBP-tagged wild type enzyme. Our results demonstrate that GyrB N-terminal arm, and specially I10 residue is essential for ATP binding/hydrolysis efficiency and DNA transfer through DNA gyrase.     

The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea

Abstract: Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5'-type I DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri , the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type I DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3'-type I DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.     

ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings

Extracts of Drosophila embryos contain an enzymatic activity that converts circular DNAs into huge networks of catenated rings in an ATP-dependent fashion. The catenation activity is resolved into two protein components during purification. One component is a novel DNA topoisomerase that requires the presence of ATP in order to relax supercoiled DNA. We have shown that the ATP-dependent DNA topoisomerase relaxes DNA by a mechanism distinct from that of nicking-closing enzymes. The Drosophila ATP-dependent topoisomerase allows one segment of a circular DNA to pass through transient breaks in both strands at another site on the DNA circle without any relative rotation between the ends at the transient break. This mechanism can convert negative supertwists to positive twists and vice versa until a relaxed equilibrium state is reached. The formation of catenated rings is mediated by an analogous bimolecular reaction which can occur between two nonhomologous DNA circles. The catenation reaction is fully reversible: in the presence of the second protein component, circular DNA is converted quantitatively into catenated forms; in its absence, the ATP-dependent topoisomerase resolves catenated networks back into monomer circles. The Drosophila ATP-dependent topoisomerase appears to be closely related to E. coli DNA gyrase in that both use a similar mechanism to change the topology of DNA, both require ATP and both are inhibited by the antibiotic novobiocin. The presence of an enzyme that allows one DNA helix to pass freely through another could not only be useful in relaxation of topological constraints, but also may be involved in the folding and unfolding of eucaryotic chromosomes.     

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.     

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.     

Studies on DNA polymerases and topoisomerases in archaebacteria

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.     

Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.