Model for the supercoiling reaction catalyzed by DNA gyrase

A hypothesis is presented which suggests that hepatitis B DNA in the Dane particles is only a partial viral genome which becomes integrated into the hepatocyte cellular DNA. The Dane particle DNA must enter a liver cell containing an active e gene, in order to become functional. It is suggested that the partial genome of hepatitis B virus is released from the cellular DNA by the mechanism of “escaping genes”.     

DNA supercoiling by gyrase is linked to nucleoid compaction

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 m cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.     

The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain.     

Nucleotide binding to DNA gyrase causes loss of DNA wrap

DNA gyrase negatively supercoils DNA in a reaction coupled to the binding and hydrolysis of ATP. Limited supercoiling can be achieved in the presence of the non-hydrolysable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (ADPNP). In order to negatively supercoil DNA, gyrase must wrap a length of DNA around itself in a positive sense. In previous work, the effect of ADPNP on the gyrase-DNA interaction has been assessed but has produced conflicting results; the aim of this work was to resolve this conflict. We have probed the wrapping of DNA around gyrase in the presence and in the absence of ADPNP using direct observation by atomic force microscopy (AFM). We confirm that gyrase indeed generates a significant curvature in DNA in the absence of nucleotide and we show that the addition of ADPNP leads to a complete loss of wrap. These results have been corroborated using a DNA relaxation assay involving topoisomerase I. We have re-analysed previous hydroxyl-radical footprinting and crystallography data, and highlight the fact that the gyrase-DNA complex is surprisingly asymmetric in the absence of nucleotide but is symmetric in the presence of ADPNP. We suggest a revised model for the conformation of DNA bound to the enzyme that is fully consistent with these AFM data, in which a closed loop of DNA is stabilised by the enzyme in the absence of ADPNP and is lost in the presence of nucleotide.     

The GyrA-box determines the geometry of DNA bound to gyrase and couples DNA binding to the nucleotide cycle

DNA gyrase catalyses the adenosine triphosphate-dependent introduction of negativesupercoils into DNA. The enzyme binds a DNA-segment at the so-called DNA-gate and cleavesboth DNA strands. DNA extending from the DNA-gate is bound at the perimeter of thecylindrical C-terminal domains (CTDs) of the GyrA subunit. The CTDs are believed tocontribute to the wrapping of DNA around gyrase in a positive node as a prerequisite forstrand passage towards negative supercoiling. A conserved seven amino acid sequence motifin the CTD, the so-called GyrA-box, has been identified as a hallmark feature of gyrases.Mutations of the GyrA-box abolish supercoiling. We show here that the GyrA-box marginallystabilizes the CTDs. Although it does not contribute to DNA binding, it is required forDNA bending and wrapping, and it determines the geometry of the bound DNA. Mutations ofthe GyrA-box abrogate a DNA-induced conformational change of the gyrase N-gate anduncouple DNA binding and adenosine triphosphate hydrolysis. Our results implicate theGyrA-box in coordinating DNA binding and the nucleotide cycle.     

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.     

Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling.

ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP. The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1. The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase. This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1. This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP. Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca. 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling. These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.     

Reverse gyrase has heat-protective DNA chaperone activity independent of supercoiling

Hyperthermophilic organisms must protect their constituent macromolecules from heat-induced degradation. A general mechanism for thermoprotection of DNA in active cells is unknown. We show that reverse gyrase, the only protein that is both specific and common to all hyperthermophiles, reduces the rate of double-stranded DNA breakage ~8-fold at 90°C. This activity does not require ATP hydrolysis and is independent of the positive supercoiling activity of the enzyme. Reverse gyrase has a minor nonspecific effect on the rate of depurination, and a major specific effect on the rate of double-strand breakage. Using electron microscopy, we show that reverse gyrase recognizes nicked DNA and recruits a protein coat to the site of damage through cooperative binding. Analogously to molecular chaperones that assist unfolded proteins, we found that reverse gyrase prevents inappropriate aggregation of denatured DNA regions and promotes correct annealing. We propose a model for a targeted protection mechanism in vivo in which reverse gyrase detects damaged DNA and acts as a molecular splint to prevent DNA breakage in the vicinity of the lesion, thus maintaining damaged DNA in a conformation that is amenable to repair.     

Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

Homology is not required for recombination mediated by DNA gyrase of Escherichia coli

Summary We have previously shown that DNA gyrase of Escherichia coli can promote recombination between heterologous DNAs in a cell-free system (Ikeda et al. 1982). In the present paper, we have studied the nucleotide sequences of several recombination junctions of -pBR322 recombinants and found that there were not more than three-basepair homologies between the parental DNAs in six combinations of the and pBR322 recombination sites. Based on this and previous results, we concluded that homology was not required for the DNA gyrase-mediated recombination. Furthermore, the structures of the recombinant DNAs we have analyzed suggest the occurrence of multiple crossovers in our in vitro system.     

Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-L-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-L-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-L-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.     

Crystal structure of reverse gyrase: insights into the positive supercoiling of DNA

Reverse gyrase is the only topoisomerase known to positively supercoil DNA. The protein appears to be unique to hyperthermophiles, where its activity is believed to protect the genome from denaturation. The 120 kDa enzyme is the only member of the type I topoisomerase family that requires ATP, which is bound and hydrolysed by a helicase-like domain. We have determined the crystal structure of reverse gyrase from Archaeoglobus fulgidus in the presence and absence of nucleotide cofactor. The structure provides the first view of an intact supercoiling enzyme, explains mechanistic differences from other type I topoisomerases and suggests a model for how the two domains of the protein cooperate to positively supercoil DNA. Coordinates have been deposited in the Protein Data Bank under accession codes 1GKU and 1GL9.     

The interaction of heat and radiation affecting the ability of nuclear DNA to undergo supercoiling changes

DNA damage (putatively strand breaks) from ionizing radiation inhibits the ability of intercalating dyes to induce right-handed supercoils in the DNA loops of HeLa nucleoids [Cook and Brazelle, J. Cell Sci. 22, 287-302 (1976); Roti Roti and Wright, Cytometry 8, 461-467 (1987)] while heat-induced changes in the nuclear matrix enhance this ability [Roti Roti and Painter, Radiat. Res. 89, 166-175 (1982)]. Since heat and radiation interact synergistically or additively on most cellular functions which they affect, the rewinding of DNA supercoils is unusual in that these agents alone affect it in an antagonistic manner. When HeLa cells were exposed to 45 degrees C for 30 min and immediately irradiated with 10 Gy of 137Cs gamma rays, the rewinding response was intermediate between that for cells which had been exposed to 10 Gy only and control. When repair of this damage was assayed in control cells, 97% of the initial damage had been repaired at 30 min postirradiation; at the same time only 10% of the initial damage had been repaired in the heat-shocked cells. This apparent dose reduction effect and the inhibition of repair were interpreted to indicate that heat-induced changes in nuclear structure were masking DNA damage from the assay and the repair system. These effects correlated with the amount of heat-induced excess protein associated with the nucleus and the nucleoid.     

Crystal structures of Thermotoga maritima reverse gyrase: inferences for the mechanism of positive DNA supercoiling

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented.     

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: II. The shape of the GyrA subunit C-terminal domain (CTD) is not a sole determinant for controlling supercoiling efficiency

DNA topoisomerases are essential enzymes that can overwind, underwind, and disentangle double-helical DNA segments to maintain the topological state of chromosomes. Nearly all bacteria utilize a unique type II topoisomerase, gyrase, which actively adds negative supercoils to chromosomes using an ATP-dependent DNA strand passage mechanism; however, the specific activities of these enzymes can vary markedly from species to species. Escherichia coli gyrase is known to favor supercoiling over decatenation (Zechiedrich, E. L., Khodursky, A. B., and Cozzarelli, N. R. (1997) Genes Dev. 11, 2580-2592), whereas the opposite has been reported for Mycobacterium tuberculosis gyrase (Aubry, A., Fisher, L. M., Jarlier, V., and Cambau, E. (2006) Biochem. Biophys. Res. Commun. 348, 158-165). Here, we set out to understand the molecular basis for these differences using structural and biochemical approaches. Contrary to expectations based on phylogenetic inferences, we find that the dedicated DNA wrapping domains (the C-terminal domains) of both gyrases are highly similar, both architecturally and in their ability to introduce writhe into DNA. However, the M. tuberculosis enzyme lacks a C-terminal control element recently uncovered in E. coli gyrase (see accompanying article (Tretter, E. M., and Berger, J. M. (2012) J. Biol. Chem. 287, 18636-18644)) and turns over ATP at a much slower rate. Together, these findings demonstrate that C-terminal domain shape is not the sole regulatory determinant of gyrase activity and instead indicate that an inability to tightly couple DNA wrapping to ATP turnover is why M. tuberculosis gyrase cannot supercoil DNA to the same extent as its γ-proteobacterial counterpart. Our observations demonstrate that gyrase has been modified in multiple ways throughout evolution to fine-tune its specific catalytic properties.     

Plasmid DNA supercoiling and gyrase activity in Escherichia coli wild-type and rpoS stationary-phase cells

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.     

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity

DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function. Removal of the CTD tail enables GyrA to introduce writhe into DNA in the absence of GyrB, an activity exhibited by other GyrA orthologs, but not by wild-type E. coli GyrA. Strikingly, a tail-less gyrase holoenzyme is markedly impaired for DNA supercoiling capacity, but displays normal ATPase function. Our findings reveal that the E. coli GyrA tail regulates DNA wrapping by the CTD to increase the coupling efficiency between ATP turnover and supercoiling, demonstrating that CTD functions can be fine-tuned to control gyrase activity in a highly sophisticated manner.     

A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

DNA gyrase is the only topoisomerase able to introduce negative supercoils into DNA. Absent in humans, gyrase is a successful target for antibacterial drugs. However, increasing drug resistance is a serious problem and new agents are urgently needed. The naturally-produced Escherichia coli toxin CcdB has been shown to target gyrase by what is predicted to be a novel mechanism. CcdB has been previously shown to stabilize the gyrase ‘cleavage complex’, but it has not been shown to inhibit the catalytic reactions of gyrase. We present data showing that CcdB does indeed inhibit the catalytic reactions of gyrase by stabilization of the cleavage complex and that the GyrA C-terminal DNA-wrapping domain and the GyrB N-terminal ATPase domain are dispensable for CcdB's action. We further investigate the role of specific GyrA residues in the action of CcdB by site-directed mutagenesis; these data corroborate a model for CcdB action based on a recent crystal structure of a CcdB–GyrA fragment complex. From this work, we are now able to present a model for CcdB action that explains all previous observations relating to CcdB–gyrase interaction. CcdB action requires a conformation of gyrase that is only revealed when DNA strand passage is taking place.