Intermediates of replication of NR1 plasmid DNA in Escherichia coli mini-cells

The pulse label of NRL plasmid-containing mini-cells has been shown to be localized mainly in DNA with a floating density in the CsCl-EtBr gradient different from the floating density of supercoil and open circle DNAs. During the chase of the pulse label, the DNA is transfered from the fraction with the intermediate floating density varying between the values for the supercoil and open circle DNA fractions to the fraction located below supercoil DNA in the equilibrium gradient and further to the open circle fraction. Electron microscopic analysis of the material with a higher floating density as compared to supercoil DNA has demonstrated the presence of "heavy" intermediates--covalently closed loosely supercoiled molecules. It is also supported by the sedimentation pattern of the characterized fraction in neutral and alkaline saccharose gradients. Molecules located in the CsCl-EtBr gradient between supercoil and open circle DNAs have the sedimentation constant characteristic for the elongation intermediates. It is suggested that NRL DNA molecules in E. coli mini-cells pass through all the basic stages of replication which results in the formation of open circle DNA or supercoil relaxation complexes.     

Quantitation of supercoiled DNA cleavage in nonradioactive DNA: application to ionizing radiation and synthetic endonuclease cleavage.

Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.     

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’.     

The role of hydroxyl radical quenching in the protection by acetate and ethylenediaminetetraacetate of supercoiled plasmid DNA from ionizing radiation-induced strand breakage

A supercoiled plasmid of 7300 base pairs was isolated and exposed in various aqueous environments to 60Co gamma-radiation. Conversion of the supercoiled form to the relaxed circular and linear forms was monitored by agarose gel electrophoresis and quantified by fluorescence scanning of the gel. Acetate, which has been reported to affect the conformation of DNA in solution, decreased the radiosensitivity of the supercoil in a concentration-dependent manner. Acetate, formate, and azide anions, as well as mannitol, all protected the supercoil from relaxation in approximate proportion to the rate at which their solutions quench the hydroxyl radical. At concentrations greater than 300 mmol dm-3, however, the efficiency of acetate radioprotection is reduced. Disodium ethylenediaminetetraacetate protected the supercoil more efficiently than would be expected from the published value of its rate constant for quenching the hydroxyl radical.     

The interaction of histones with simian virus 40 supercoiled circular deoxyribonucleic acid in vitro.

The interaction of supercoiled, circular SV40 DNA with calf thymus histone fractions has been studied. Five- to ten-fold less f1 histone is required to complex a given amount of DNA compared to the other histones. When the supercoiled DNA is converted to either the relaxed circular form, or full length linear molecules, or gragmented linear or denatured stands, the efficiency of complex formation with f1 histone markedly decreases. We conclude that f1 histone has a special ability to interact with supercoiled DNA. This conclusion is supported by the fact that supercoiled circular Col E1 DNA interacts with f1 as efficiently as does SV40 DNA.     

Insertion of L1 elements into sites that can form non-B DNA. Interactions of non-B DNA-forming sequences

Three rat L1 element integration (target) sites chosen at random can adopt non-B DNA structures in vitro at normal bacterial superhelical densities. These target sites contain, respectively, short, mixed (AT)n tracts that we show can form one or more cruciforms, short (GT)n tracts, or polypurine:polypyrimidine regions. These sites share no sequence homology, and a non-B DNA structure appears to be the only feature common to them all. When the right end of the L1Rn3 element which forms a complex series of non-B DNA structures including two triplexes, and its target site which undergoes cruciform extrusion, are present on the same supercoiled molecule, they compete for available supercoil energy. The amount of non-B DNA formed at each site varies with pH, the concentration of cations, and the size of the topological domain. The implication of our findings for recombination of L1 elements and for the effect of these elements on contiguous DNA sequences is discussed.     

Characterization of recombinant human DNA topoisomerase IIIalpha activity expressed in yeast

Recombinant human DNA topoisomerase IIIalpha was expressed in mutant yeast cells devoid of both topoisomerases I and III, and the gene product was partially purified. The activity of the protein in supercoil removal was found to be limited and also biphasic: in the first phase it processively changed the linking-number of hypernegatively supercoiled DNA, but only to the superhelicity of a regular negative supercoil; in the second phase the enzyme relaxed the DNA further, but only slightly and slowly. The optimal solution conditions for the enzyme activity were found to be physiological. The assay results with a truncation mutant showed that the C-terminal 334 amino acids are unnecessary for the activity, suggesting that this region, and perhaps the entire protein, is involved in a function other than supercoil removal.     

Isolation,characterization, and structure of the folded interphase genome of Drosophila melanogaster

The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% Nonidet P40. The nonviscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranucleosome histones: H2A, H2B, H3, and H4).The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs—that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26°C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres.Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA.The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA-DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360° left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.     

The effect of supercoil and temperature on the recognition of palindromic and non-palindromic regions in phi X174 replicative form DNA by S1 and Bal31

The effect of supercoil and temperature on the topology of phi X174 replicative form (RF) DNA was studied using single-strand specific endonucleases S1 and Bal31 as probes for cruciform extrusion and other structural perturbations of the B-helix. Both enzymes were found to recognize specifically and reproducibly over 30 sites, most of which were cleaved by both enzymes independent of the superhelicity of the genome. A negative superhelical density exceeding 0.06 stabilized a transition in the DNA conformation that generated several new cleavage sites for Bal31. The underlying structures appeared to be only transiently stable and were lost from in vitro supercoiled DNA during brief incubation at 65 degrees C. They were generally absent from in vivo supercoiled RF DNA of equal superhelicity as a consequence of the extraction and storage procedure. Mapping of the cleavage sites suggested that they were preferentially located near the beginnings and ends of genes and that the structural basis for at least some of them was the extrusion of relatively small palindromes into the cruciform state. Insertion of a short synthetic palindromic sequence into the phi X174 genome generated a supercoil-dependent, temperature-sensitive secondary structure that was cleaved in the Bal31 but not the S1 reaction, further supporting the hypothesis that even small cruciforms with stem size of 7 or less base pairs may be transiently stable. Subjecting supercoiled RF DNA to the typical S1 reaction conditions induced a topological shift that diminished all but one of the supercoil-induced Bal31 recognition sites and promoted the formation of one major new site.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.     

Statistical mechanical theory of melting transition in supercoiled DNA

The melting curve for a covalently closed circular DNA has been analyzed on the basis of an expression for the supercoiling energy derived in terms of the elastic parameters of the macromolecule, treated as a homopolymer. The result obtained by applying the usual methods of statistical mechanics indicate close agreement with the available experimental data. It is found that the elevation of the melting temperature, as compared to that of the nicked circular or linear DNA, is a natural consequence of the fact that the supercoiled molecule is constrained by an invariant linking number. The flattening of the melting curve, on the other hand, arises as the closed circular duplex melts into a loose helix rather than random coils.     

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.     

Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA

High mobility group (HMG) 1 protein and its two homologous DNA-binding domains, A and B ("HMG-boxes"), can bend and supercoil DNA in the presence of topoisomerase I, as well as recognize differently bent and distorted DNA structures, including four-way DNA junctions, supercoiled DNA and DNA modified with anticancer drug cisplatin. Here we show that the lysine-rich part of the linker region between A and B domains of HMG-1, the (85)TKKKFKD(91) sequence that is attached to the N-terminus of the B domain within HMG-1, is a prerequisite for a preferential binding of the B domain to supercoiled DNA. The above sequence is also essential for a high-affinity binding of the B domain to DNA containing a site-specific major 1,2-d(GpG) intrastrand DNA adduct of cisplatin. Mutation of Arg(97), but not Lys(90) [Lys(90) forms a specific cross-link with platinum(II) in major groove of cisplatin-modified DNA; Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180--2188], to alanine significantly (>40-fold) reduces affinity of the B domain to cisplatin-modified DNA, inhibits the ability of the B domain to bend (ligase-mediated circularization) or supercoil DNA, and results in a loss of the preferential binding of the B domain to supercoiled DNA without affecting the structural-specificity of the HMG-box for four-way DNA junctions. Some of the reported activities of the B domain are enhanced when the B domain is covalently linked to the A domain. We propose that binding of the A/B linker region within the major DNA groove helps the two HMG-1 domains to anchor to the minor DNA groove to facilitate their DNA binding and other activities.     

The effects of deoxyribonucleic acid secondary structure on tertiary structure.

The secondary structure of supercoiled DNA was varied by changes in ionic strength. For I = 0.075-0.4 the structure remained in the previously established branched form with only minor alterations in molecular dimensions. In 4M-NaCl, which induces linear DNA to change its secondary structure to the C structure and brings about an increase in the superhelix density of the molecule, no extra branches were observed on the molecules. The limiting factors that dictate supercoil structure seem to be the number and position of potential branch points and the proximity with which the two intertwining DNA strands can approach each other on the arms of the branches. This value is close to 10nm under the conditions described, and is 14-15nm at I = 0.2. It is suggested that such values should be borne in mind when models of chromosome structure are being constructed.     

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.     

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.     

Effect of supercoiling on the melting characteristics of heteropolynucleotides

Considering a supercoiled DNA molecule, having equal numbers of two distinct types of base-pairs, it has been shown theoretically that even for the extreme cases of mixing of the two types of base-pairs in a supercoiled DNA, the melting temperatures as well as the melting curves do not differ significantly. This indicates that these properties are practically independent of the detailed base sequence when the molecule is a covalently closed one and may be replaced by an equivalent homopolynucleotide whose binding energy is equal to the average base-pairing energy of the original DNA. This conclusion has been further supported by comparing the theoretical results with those obtained experimentally in the cases of polyoma DNA and phi X174 DNA. Finally, the effects of supercoiling on the cooperativity of melting and a few aspects of the differential melting characteristics of a supercoiled DNA have been discussed which provide a clear physical understanding of the process.