DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.     

Negative supercoiling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate.

In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.     

Positive supercoiling catalysed in vitro by ATP-dependent topoisomerase from Desulfurococcus amylolyticus

A topoisomerase capable of introducing positive supercoils into closed-circular DNA has been isolated from the extremely thermophilic anaerobic archaebacterium Desulfurococcus amylolyticus. This polypeptide has an Mr of 135,000, as determined by electrophoresis under denaturing conditions. The enzyme is active in the temperature range from 65 degrees C to 100 degrees C and catalyzes positive supercoiling both in negatively supercoiled DNA and in relaxed DNA. These reactions require the presence of ATP. The enzyme's action on a single topoisomer has shown the linking number to increase by an integral number upon the relaxation of negative supercoils and the introduction of positive ones. This means that the reverse gyrase from D. amylolyticus is a type I topoisomerase. The presence of an extended AT sequence within the closed-circular DNA enhances the activity of the Desulfurococcus topoisomerase. Even though the enzyme is isolated from a strictly anaerobic bacterium, it is fully active in the presence of oxygen.     

Adenosine 5'-O-(3-thio)triphosphate (ATPgammaS) promotes positive supercoiling of DNA by T. maritima reverse gyrase

Reverse gyrases are topoisomerases that catalyze ATP-dependent positive supercoiling of circular covalently closed DNA. They consist of an N-terminal helicase-like domain, fused to a C-terminal topoisomerase I-like domain. Most of our knowledge on reverse gyrase-mediated positive DNA supercoiling is based on studies of archaeal enzymes. To identify general and individual properties of reverse gyrases, we set out to characterize the reverse gyrase from a hyperthermophilic eubacterium. Thermotoga maritima reverse gyrase relaxes negatively supercoiled DNA in the presence of ADP or the non-hydrolyzable ATP-analog ADPNP. Nucleotide binding is necessary, but not sufficient for the relaxation reaction. In the presence of ATP, positive supercoils are introduced at temperatures above 50 degrees C. However, ATP hydrolysis is stimulated by DNA already at 37 degrees C, suggesting that reverse gyrase is not frozen at this temperature, but capable of undergoing inter-domain communication. Positive supercoiling by reverse gyrase is strictly coupled to ATP hydrolysis. At the physiological temperature of 75 degrees C, reverse gyrase binds and hydrolyzes ATPgammaS. Surprisingly, ATPgammaS hydrolysis is stimulated by DNA, and efficiently promotes positive DNA supercoiling, demonstrating that inter-domain communication during positive supercoiling is fully functional with both ATP and ATPgammaS. These findings support a model for communication between helicase-like and topoisomerase domains in reverse gyrase, in which an ATP and DNA-induced closure of the cleft in the helicase-like domain initiates a cycle of conformational changes that leads to positive DNA supercoiling.     

Human topoisomerase IIalpha rapidly relaxes positively supercoiled DNA: implications for enzyme action ahead of replication forks

Movement of the DNA replication machinery through the double helix induces acute positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single- and double-stranded DNA breaks, respectively, it has been assumed that type I enzymes relax the positive supercoils that precede the replication fork. Conversely, type II enzymes primarily resolve the precatenanes and untangle catenated daughter chromosomes. However, studies on yeast and bacteria suggest that type II topoisomerases may also function ahead of the replication machinery. If this is the case, then positive DNA supercoils should be the preferred relaxation substrate for topoisomerase IIalpha, the enzyme isoform involved in replicative processes in humans. Results indicate that human topoisomerase IIalpha relaxes positively supercoiled plasmids >10-fold faster than negatively supercoiled molecules. In contrast, topoisomerase IIbeta, which is not required for DNA replication, displays no such preference. In addition to its high rates of relaxation, topoisomerase IIalpha maintains lower levels of DNA cleavage complexes with positively supercoiled molecules. These properties suggest that human topoisomerase IIalpha has the potential to alleviate torsional stress ahead of replication forks in an efficient and safe manner.     

Identification of a DNA supercoiling activity in Saccharomyces cerevisiae.

A relaxed plasmid DNA is shown to become positively supercoiled in cell extracts from top1 strains of Saccharomyces cerevisiae. This positive supercoiling activity is dependent on the presence of bacterial DNA topoisomerase I and ATP (or dATP), and the positive supercoils generated in this reaction are not constrained by protein(s). Non-hydrolyzable ATP analogs cannot substitute for ATP in this supercoiling reaction, and the supercoiling activity is not due to RNA synthesis. The presence of an ARS sequence in the DNA does not alter the activity. Furthermore, this activity is equally active against UV irradiated or intact DNA. Extracts prepared from rad50 and rad52 mutant cells exhibited the same activity. Partial purification of this activity suggests that a protein factor with a native molecular weight of approximately 150 kDa is primarily responsible for the activity. The possibility that this supercoiling activity may be due to tracking of a protein along the intact duplex DNA is discussed.     

Intrinsic DNA-dependent ATPase activity of reverse gyrase

Reverse gyrase is a type I DNA topoisomerase that promotes positive supercoiling of closed-circular double-stranded DNA through an ATP-dependent reaction, and it was purified from an archaebacterium, Sulfolobus. When ATP is replaced by UTP, GTP, or CTP, this enzyme just relaxes the negatively supercoiled closed-circular double-stranded DNA. We found that reverse gyrase hydrolyzes ATP through a double-stranded DNA-dependent reaction. The superhelicity of the DNA did not affect the ATPase activity. However, reverse gyrase does not hydrolyze UTP, GTP, or CTP. Therefore, any of the four nucleotide 5'-triphosphates acts as an effector for the topoisomerase activity of reverse gyrase, but only ATP supports the positive supercoiling of closed-circular double-stranded DNA, through the energy released on its hydrolysis. Single-stranded DNA was a much more potent cofactor for the ATPase activity of the enzyme than double-stranded DNA, and it acted as a potent inhibitor for the topoisomerase activity on double-stranded DNA. These results indicate that reverse gyrase has higher affinity to single-stranded DNA than to double-stranded DNA, which suggests a cellular function of the enzyme.     

Varying levels of positive and negative supercoiling differently affect the efficiency with which topoisomerase II catenates and decatenates DNA

Type II DNA topoisomerases catalyze the transport of one DNA double helix through another. Here, by using a non-hydrolyzable analog of ATP, I examined the single-step DNA transport preferences of the yeast type II topoisomerase bound to positively and negatively supercoiled DNA rings. I found that negative supercoiling favors decatenation of DNA rings more than positive supercoiling. Conversely, positive supercoiling favors the catenation and knotting of DNA rings more than negative supercoiling. This vectorial effect of DNA supercoiling handedness supports a model in which type II topoisomerases can recognize three DNA segments, and highlights a novel influence of DNA supercoiling in global DNA topology.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

Nucleotide- and stoichiometry-dependent DNA supercoiling by reverse gyrase

Reverse gyrase is a unique type IA topoisomerase that can introduce positive supercoils into DNA. We have investigated some of the biochemical properties of Archaeoglobus fulgidus reverse gyrase. It can mediate three distinct supercoiling reactions depending on the adenine nucleotide cofactor that is present in the reaction. Besides the ATP-driven positive supercoiling reaction, the enzyme can introduce negative supercoils with a nonhydrolyzable analog, adenylyl imidodiphosphate. In the presence of ADP the plasmid DNA is relaxed almost completely, leaving a very low level of positive supercoiling. Surprisingly, the final supercoiling extent for all three distinct reactions depends on the stoichiometry of enzyme to DNA. This dependence is not due to the difference of reaction rate, suggesting that the amount of enzyme bound to DNA is an important determinant for the final supercoiling state of the reaction product. Reverse gyrase also displays exquisite sensitivity toward temperature. Raising the reaction temperatures from 80 to 85 degrees C, both of which are within the optimal growth temperature of A. fulgidus, greatly increases enzyme activity for all the supercoiling reactions. For the reaction with AMPPNP, the product is a hypernegatively supercoiled DNA. This dramatic enhancement of the reverse gyrase activity is also correlated with the appearance of DNA in a pre-melting state at 85 degrees C, likely due to the presence of extensively unwound regions in the plasmid. The possible mechanistic insights from these findings will be presented here.     

DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.     

An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity

Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli, typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP-dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.     

Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.     

Reverse gyrase binding to DNA alters the double helix structure and produces single-strand cleavage in the absence of ATP.

Stoichiometric amounts of pure reverse gyrase, a type I topoisomerase from the archaebacterium Sulfolobus acidocaldarius were incubated at 75 degrees C with circular DNA containing a single-chain scission. After covalent closure by a thermophilic ligase and removal of bound protein molecules, negatively supercoiled DNA was produced. This finding, obtained in the absence of ATP, contrasts with the ATP-dependent positive supercoiling catalyzed by reverse gyrase and is interpreted as the result of enzyme binding to DNA at high temperature. Another consequence of reverse gyrase stoichiometric binding to DNA is the formation of a cleavable complex which results in the production of single-strand breaks in the presence of detergent. Like eubacterial type I topoisomerase (protein omega), reverse gyrase is tightly attached to the 5' termini of the cleaved DNA. In the light of these results, a comparison is tentatively made between reverse gyrase and the eubacterial type I (omega) and type II (gyrase) topoisomerases.     

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.     

Ability of viral topoisomerase II to discern the handedness of supercoiled DNA: bimodal recognition of DNA geometry by type II enzymes

Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIalpha and Escherichia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase IIalpha and topoisomerase IIbeta to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under- and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domain during DNA cleavage.