How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation

Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation.     

Duplex DNA knots produced by Escherichia coli topoisomerase I. Structure and requirements for formation

We investigated systematically the knotting of nicked circular duplex DNA by Escherichia coli topoisomerase I. Agarose gel electrophoresis of knots forms a ladder of DNA bands. Each rung is made up of a variety of knots with the same number of nodes, or segment crossings; knots in adjacent rungs differ by one node. We extended the technique of electron microscopy of recA protein-coated DNA to the visualization of the complex knots tied by topoisomerase I. The striking result is that the enzyme produces every knot theoretically possible. The requirement for excess enzyme to form complex knots suggests a role for topoisomerase I in contorting the DNA in addition to promoting strand passage. We conclude that nodes formed are equally likely to be positive or negative and that topoisomerase I can pass DNA strands through a transient enzyme-generated break without regard to orientation of the passing strand. The results are interpreted in terms of a formulation for the topological requirements for knotting.     

The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant.

Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed.     

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation.     

DNA supercoiling inhibits DNA knotting

Despite the fact that in living cells DNA molecules are long and highly crowded, they are rarely knotted. DNA knotting interferes with the normal functioning of the DNA and, therefore, molecular mechanisms evolved that maintain the knotting and catenation level below that which would be achieved if the DNA segments could pass randomly through each other. Biochemical experiments with torsionally relaxed DNA demonstrated earlier that type II DNA topoisomerases that permit inter- and intramolecular passages between segments of DNA molecules use the energy of ATP hydrolysis to select passages that lead to unknotting rather than to the formation of knots. Using numerical simulations, we identify here another mechanism by which topoisomerases can keep the knotting level low. We observe that DNA supercoiling, such as found in bacterial cells, creates a situation where intramolecular passages leading to knotting are opposed by the free-energy change connected to transitions from unknotted to knotted circular DNA molecules.     

Description of the topological entanglement of DNA catenanes and knots by a powerful method involving strand passage and recombination

We utilize a recently discovered, powerful method to classify the topological state of knots and catenanes. In this method, each such form is associated with a unique polynomial. These polynomials allow a rigorous determination of whether knotted or catenated DNA molecules that appear distinct actually are, and indicate the structure of related molecules. A tabulation is given of the polynomials for all possible stereoisomers of many of the knotted and catenated forms that are found in DNA. The polynomials for a substrate DNA molecule and the products obtained from it by either recombination or strand passage by a topoisomerase are related by a simple theorem. This theorem affords natural applications of the polynomial method to these processes. Examples are presented involving site-specific recombination by the transposon Tn3-encoded resolvase and the phage lambda integrase, in which product structure is predicted as a function of crossover mechanism.     

Knotting of DNA molecules isolated from deletion mutants of intact bacteriophage P4.

DNA molecules isolated from tailless phage particles (capsids) of bacteriophage P4 virl del10 are known to be knotted. We have found by electron microscopy that 80% of DNA molecules isolated from intact phage particles of P4 virl del10 also contained knots. This observation indicates that the predominant form of P4 virl del10 DNA within the intact phage particle is either knotted or in a configuration that permits knotting upon isolation. In comparison to P4 virl del10 (deleted 1000 basepairs), DNA molecules isolated from intact P4 virl del2 (deleted 650 basepairs) and P4 virl (non-deleted) contained 50% and 15% knots respectively, showing an association of decreased size of deletion of DNA with a decreased fraction of knotted genomes.     

Knotting dynamics during DNA replication

The topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two-dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.     

Interlocking and knotting of ring nucleoli in amphibian oocytes

Various configurations of interlocking and knotting of ring nucleoli from amphibian oocytes have been identified in material taken from 2 females of Eurycea bislineata and one female of Plethodon cinereus. The simplest configuration is a simple interlock between 2 rings of similar or different lengths. More complex interlocks have been seen, in which 3 rings are linked together in such a way that they cannot be extended into a chain. A third configuration involves complex knotting of single long rings. It is suggested that the interlocked configurations arise through the replication of rings of DNA that have low levels of supercoiling, and that the knotted rings arise by misjoining of the ends of linear molecules that have become wound around one another. Models are suggested in support of these proposals and the significance of interlocking of ring nucleoli in relation to the mechanism of gene amplification is discussed.     

Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.     

Geometric arrangements of Tn3 resolvase sites

Site-specific recombination by Tn3 resolvase normally occurs in vitro and in vivo only between directly repeated res sites on the same supercoiled DNA molecule. However, with multiply interlinked catenane substrates consisting of two DNA rings each containing a single res site, resolvase efficiently carried out intermolecular recombination. The topology of the knots produced by several rounds of this reaction proves that the DNA within the synaptic intermediate is coiled in an interwound (plectonemic) fashion rather than wrapped solenoidally around resolvase as in previously characterized supercoiled DNA-protein complexes. The synaptic intermediate can contain equivalently supercoil, catenane, or knot crossings as long as the res sites have a right-handed coiling and a particular relative orientation. The structure of the product knots and catenanes also shows the path the DNA takes during strand exchange. Intermolecular recombination within multiply linked catenanes required negative supercoiling, as does the standard intramolecular reaction.     

Production of highly knotted DNA by means of cosmid circularization inside phage capsids

Background  

The formation of DNA knots is common during biological transactions. Yet, functional implications of knotted DNA are not fully understood. Moreover, potential applications of DNA molecules condensed by means of knotting remain to be explored. A convenient method to produce abundant highly knotted DNA would be highly valuable for these studies.     

Underwinding of DNA associated with duplex-duplex pairing by RecA protein

Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.     

In vitro catenation and decatenation of DNA and a novel eucaryotic ATP-dependent topoisomerase

Extracts from X. laevis germinal vesicles interlock duplex DNA circles to form catenanes. The catenation activity requires Mg++ and ATP. Negatively supercoiled or relaxed DNA can be used as substrates for the catenation reaction. Homology between donor and acceptor DNA is not required, since catenanes are formed between DNA molecules with unrelated sequences. In the course of the isolation of the activity responsible for the catenation reaction, we discovered a new ATP-dependent topoisomerase. The fractions containing the novel topoisomerase catenate and decatenate DNA, the ionic strength dictating which of the two opposing reactions will occur.     

Electrophoretic mobility of supercoiled,catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.     

Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases.

DNA molecules isolated from bacteriophage P4 are mostly linear with cohesive ends capable of forming circular and concatemeric structures. In contrast, almost all DNA molecules isolated form P4 tailless capsids (heads) are monomeric DNA circles with their cohesive ends hydrogen-bonded. Different form simple DNA circles, such P4 head DNA circles contain topological knots. Gel electrophoretic and electronmicroscopic analyses of P4 head DNA indicate that the topological knots are highly complex and heterogeneous. Resolution of such complex knots has been studied with various DNA topoisomerases. The conversion of highly knotted P4 DNA to its simple circular form is demonstrated by type II DNA topoisomerases which catalyze the topological passing of two crossing double-stranded DNA segments [Liu, L. F., Liu, C. C. & Alberts, B. M. (1980) Cell, 19, 697-707]. The knotted P4 head DNA can be used in a sensitive assay for the detection of a type II DNA topoisomerase even in the presence of excess type I DNA topoisomerases.     

Novel display of knotted DNA molecules by two-dimensional gel electrophoresis

We describe a two-dimensional agarose gel electrophoresis procedure that improves the resolution of knotted DNA molecules. The first gel dimension is run at low voltage, and DNA knots migrate according to their compactness. The second gel dimension is run at high voltage, and DNA knots migrate according to other physical parameters such as shape and flexibility. In comparison with one-dimensional gel electrophoresis, this procedure segregates the knotted DNA molecules from other unknotted forms of DNA, and partially resolves populations of knots that have the same number of crossings. The two-dimensional display may allow quantitative and qualitative characterization of different types of DNA knots simply by gel velocity.     

Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.     

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.     

Inhibition of Zn(II) Binding Type IA Topoisomerases by Organomercury Compounds and Hg(II)

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.