The structural basis for substrate specificity in DNA topoisomerase IV

Most bacteria possess two type IIA topoisomerases, DNA gyrase and topo IV, that together help manage chromosome integrity and topology. Gyrase primarily introduces negative supercoils into DNA, an activity mediated by the C-terminal domain of its DNA binding subunit (GyrA). Although closely related to gyrase, topo IV preferentially decatenates DNA and relaxes positive supercoils. Here we report the structure of the full-length Escherichia coli ParC dimer at 3.0 A resolution. The N-terminal DNA binding region of ParC is highly similar to that of GyrA, but the ParC dimer adopts a markedly different conformation. The C-terminal domain (CTD) of ParC is revealed to be a degenerate form of the homologous GyrA CTD, and is anchored to the top of the N-terminal domains in a configuration different from that thought to occur in gyrase. Biochemical assays show that the ParC CTD controls the substrate specificity of topo IV, likely by capturing DNA segments of certain crossover geometries. This work delineates strong mechanistic parallels between topo IV and gyrase, while explaining how structural differences between the two enzyme families have led to distinct activity profiles. These findings in turn explain how the structures and functions of bacterial type IIA topoisomerases have evolved to meet specific needs of different bacterial families for the control of chromosome superstructure.     

Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque

E. coli DNA gyrase uses the energy of ATP hydrolysis to introduce essential negative supercoils into the genome, thereby working against the mechanical stresses that accumulate in supercoiled DNA. Using a magnetic-tweezers assay, we demonstrate that small changes in force and torque can switch gyrase among three distinct modes of activity. Under low mechanical stress, gyrase introduces negative supercoils by a mechanism that depends on DNA wrapping. Elevated tension or positive torque suppresses DNA wrapping, revealing a second mode of activity that resembles the activity of topoisomerase IV. This 'distal T-segment capture' mode results in active relaxation of left-handed braids and positive supercoils. A third mode is responsible for the ATP-independent relaxation of negative supercoils. We present a branched kinetic model that quantitatively accounts for all of our single-molecule results and agrees with existing biochemical data.     

Structural basis for the MukB‐topoisomerase IV interaction and its functional implications in vivo

Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB–topo IV complex to 2.3 Å resolution. The structure shows that the so‐called ‘hinge’ region of MukB forms a heterotetrameric assembly with a C‐terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement.     

Topoisomerase IV bends and overtwists DNA upon binding

Escherichia coli topoisomerase IV (Topo IV) is an essential ATP-dependent enzyme that unlinks sister chromosomes during replication and efficiently removes positive but not negative supercoils. In this article, we investigate the binding properties of Topo IV onto DNA in the absence of ATP using a single molecule micromanipulation setup. We find that the enzyme binds cooperatively (Hill coefficient alpha approximately 4) with supercoiled DNA, suggesting that the Topo IV subunits assemble upon binding onto DNA. It interacts preferentially with (+) rather than (-) supercoiled DNA (Kd+=0.15 nM, Kd-=0.23 nM) and more than two orders-of-magnitude more weakly with relaxed DNA (Kd0 approximately 36 nM). Like gyrase but unlike the eukaryotic Topo II, Topo IV bends DNA with a radius 0= 6.4 nm and locally changes its twist and/or its writhe by 0.16 turn per bound complex. We estimate its free energy of binding and study the dynamics of interaction of Topo IV with DNA at the binding threshold. We find that the protein/DNA complex alternates between two states: a weakly bound state where it stays with probability p = 0.89 and a strongly bound state (with probability p = 0.11). The methodology introduced here to characterize the Topo IV/DNA complex is very general and could be used to study other DNA/protein complexes.     

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.     

The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication.

Mutants in bacterial topoisomerase (topo) IV are deficient in chromosomal partitioning. To investigate the basis of this phenotype, we examined plasmid DNA topology in conditionally lethal topo IV mutants. We found that dimeric catenated plasmids accumulated in vivo after topo IV inhibition. The catenanes were supercoiled, contained from 2 to > 32 nodes, and were the products of DNA synthesis. Electron microscopy and recombination tests proved that the catenanes have the unique structure predicted for replication intermediates. These data provide strong evidence for a model in which unlinking of the double helix can occur in two stages during DNA replication and for the critical role of topo IV in the second stage. The interlocks in the catenanes appear to be sequestered from DNA gyrase, perhaps by compartmentalization in an enzyme complex dedicated to partitioning.     

CK2-Mediated Hyperphosphorylation of Topoisomerase I Targets Serine 506, Enhances Topoisomerase I–DNA Binding,and Increases Cellular Camptothecin Sensitivity

Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies.     

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.     

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.     

Binding of two DNA molecules by type II topoisomerases for decatenation

Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation.     

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as ‘topological poisons of topoisomerase I’.     

High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=     

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.     

Reverse gyrase functions as a DNA renaturase: annealing of complementary single-stranded circles and positive supercoiling of a bubble substrate

Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.     

Unlinking of supercoiled DNA catenanes by type IIA topoisomerases

It was found recently that DNA catenanes, formed during replication of circular plasmids, become positively (+) supercoiled, and the unlinking of such catenanes by type IIA topoisomerases proceeds much more efficiently than the unlinking of negatively (−) supercoiled catenanes. In an attempt to explain this striking finding we studied, by computer simulation, conformational properties of supercoiled DNA catenanes. Although the simulation showed that conformational properties of (+) and (−) supercoiled replication catenanes are very different, these properties per se do not give any advantage to (+) supercoiled over (−) supercoiled DNA catenanes for unlinking. An advantage became evident, however, when we took into account the established features of the enzymatic reaction catalyzed by the topoisomerases. The enzymes create a sharp DNA bend in the first bound DNA segment and allow for the transport of the second segment only from inside the bend to its outside. We showed that in (−) supercoiled DNA catenanes this protein-bound bent segment becomes nearly inaccessible for segments of the other linked DNA molecule, inhibiting the unlinking.     

Drosophila melanogaster topoisomerase IIIalpha preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development

Eukaryotic type IA topoisomerases are important for the normal function of the cell, and in some cases essential for the organism, although their role in DNA metabolism remains to be elucidated. In this study, we cloned Drosophila melanogaster topoisomerase (topo) IIIalpha from an embryonic cDNA library and expressed and purified the protein to >95% homogeneity. This enzyme partially relaxes a hypernegatively supercoiled plasmid substrate consistent with other purified topo IIIs. A novel, covalently closed bubble substrate was prepared for this study, which topo IIIalpha fully relaxed, regardless of the handedness of the supercoils. Experiments with the bubble substrate demonstrate that topo IIIalpha has much different reaction preferences from those obtained by plasmid substrate-based assays. This is presumably due to the fact that solution conditions can affect the structure of plasmid based substrates and therefore their suitability as a substrate. A mutant allele of the Top3alpha gene, Top3alpha191, was isolated through imprecise excision mutagenesis of an existing P-element inserted in the first intron of the gene. Top3alpha191 is recessive lethal, with most of the homozygous individuals surviving to pupation but never emerging to adulthood. Whereas this mutation can be rescued by a Top3alpha transgene, ubiquitous overexpression of D. melanogaster topo IIIbeta cannot rescue this allele.