Helix stability and the mechanism of cruciform extrusion in supercoiled DNA molecules

The kinetic properties of cruciform extrusion in supercoiled DNA molecules fall into two main classes. C-type cruciforms extrude in the absence of added salt, at relatively low temperatures, with large activation energies, while S-type cruciforms exhibit no extrusion in the absence of salt, and maximal rates at 50 mM NaCl, with activation energies about one quarter those of the C-type. These diverse properties are believed to reflect two distinct pathways for the extrusion process, and are determined by the nature of the sequences which form the context of the inverted repeat. C-type kinetics are conferred by A + T rich sequences, implying a role of helix stability in the selection. In this study we have shown that: 1. Helix-destabilising solvents (dimethyl formamide and formamide) facilitate extrusion by normally S-type molecules at low temperatures in the absence of salt. 2. C-type extrusion is strongly suppressed by low concentrations (2-4 microM) distamycin, at which concentrations S-type extrusion is enhanced. 3. Some extrusion occurs in a C-type construct in the presence of 50 mM NaCl. This is increased by addition of 3 microM distamycin, under which conditions extrusion becomes effectively S-type. Thus S-type constructs can behave in a quasi-C-type manner in the presence of helix-destabilising solvents, and C-type extrusion is suppressed by binding a compound which stabilises A + T rich regions of DNA. Helix destabilisation leads to C-type behaviour, while helix stabilisation results in S-type properties. These studies demonstrate the influence of contextual helix stability on the selection of kinetic mechanism of cruciform extrusion.     

Localized chemical hyperreactivity in supercoiled DNA: evidence for base unpairing in sequences that induce low-salt cruciform extrusion

Certain A + T-rich DNA sequences (C-type inducing sequences) cause adjacent inverted repeats to undergo cruciform extrusion by a particular pathway (C-type extrusion), which is characterized by large activation energies and extrusion at low salt concentrations and relatively low temperatures. When they are supercoiled, these sequences become reactive toward the normally single-strand-selective reagents bromoacetaldehyde, glyoxal, osmium tetraoxide, and sodium bisulfite. The following evidence is presented: (1) The most reactive sequences are those to the left of the inverted repeat. (2) Chemical reactivity is suppressed by either sodium chloride or micromolar concentrations of distamycin. The suppression of reactivity closely parallels that of C-type cruciform extrusion. (3) Chemical reactivity requires a threshold level of negative supercoiling. The threshold superhelix density depends on the prevailing salt concentration. (4) Analysis of temperature dependences suggests that reaction with osmium tetraoxide involves transient unstacking events, while bromoacetaldehyde requires larger scale helix opening. Thus a variety of opening events may occur in the supercoiled A + T-rich sequences, from small-amplitude breathing to low-frequency, large-amplitude openings. The latter appear to be responsible for C-type cruciform extrusion.     

Large-scale stable opening of supercoiled DNA in response to temperature and supercoiling in (A + T)-rich regions that promote low-salt cruciform extrusion.

We have studied the properties of (A + T)-rich sequences derived from ColE1 that promote cruciform extrusion at low ionic strength in supercoiled plasmids. We compared the chemical reactivity of the sequences in negatively supercoiled DNA (using osmium tetroxide and bromoacetaldehyde) with the results of two-dimensional gel electrophoresis performed under the same conditions. Taken together, the results indicate the occurrence of cooperative helix-coil transitions in the (A + T)-rich DNA at low ionic strength, to form stable, denatured regions. The extent of the open region is a function of temperature and superhelix density, with an additional local destabilization brought about by the presence of cruciform structures. We present a simple statistical mechanical model of the helix-coil transition in the (A + T)-rich DNA, from which we have obtained estimates of the free energy for average base-pair opening of 0.31 kcal mol-1 and that for the formation of a helix-coil junction of 4.9 kcal mol-1, in 45 mM Tris-borate, pH 8.3, 0.5 mM EDTA. The results offer a model for the C-type mechanism of cruciform extrusion. Inverted repeats that are incorporated into the melted region undergo hairpin loop formation below 50 degrees C, and upon closure of the melted region, by reduction of temperature or increased ionic strength, they remain as a fully extruded cruciform structure.     

The kinetic properties of cruciform extrusion are determined by DNA base-sequence.

The extrusion kinetics of two cruciforms derived from unrelated DNA sequences differ markedly. Kinetic barriers exist for both reactions, necessitating elevated temperatures before extrusion proceeds at measureable speeds, but the dependence upon temperature and ionic strength is quite different for the two sequences. One, the ColE1 inverted repeat, exhibits a remarkably great temperature dependence of reaction rate and is suppressed by moderate amounts of NaCl or MgCl2. In contrast, the other, a synthetic inverted repeat present in pIRbke8, shows more modest temperature dependence and has a requirement for the presence of salt, with optimal concentrations being 50 mM NaCl or 100 microM MgCl2. Under optimal conditions, cruciform extrusion rates are fast (t1/2 less than 60m) at 37 degrees C for both sequences at native superhelix densities. In 50 mM NaCl the pIRbke8 inverted repeat is characterised by an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole -1. The differences in kinetic properties between the two sequences indicate that DNA base sequence is itself an important factor in determining cruciform kinetics, and possibly even in the selection of the mechanistic pathway.     

Structure- and sequence-specificity of ozone degradation of supercoiled plasmid DNA.

Ozone-reactive sites on the nucleobase moieties in supercoiled pBR322 DNA were investigated by using sequencing procedures. Ozonolysis in the absence of salt resulted in degradation of thymine residues in the A + T rich region located at 3100-3400bp. In the presence of salt, such as NaCl or MgCl2, a conformational change of plasmid DNA was induced. Subsequently the thymine and guanine residues in the loop of the cruciform located at 3120bp and 3220bp were degraded. In addition, central thymine residues present in sequences GTA, GTT and ATA were also degraded.     

Long range structural communication between sequences in supercoiled DNA. Sequence dependence of contextual influence on cruciform extrusion mechanism

Sequence context may profoundly alter the character of structural transitions in supercoiled DNA (Sullivan, K. M., and Lilley, D. M. J. (1986) Cell 47, 817-827). The A + T-rich sequences of ColE1, which flank the inverted repeat, are responsible for cruciform extrusion following a mechanistic pathway which proceeds via a relatively large denatured region. This C-type mechanism results in kinetic properties which are very different from those of the S-type pathway, the normal mechanism of cruciform extrusion in the absence of the ColE1 flanking sequences. We have analyzed the sequence requirements for the induction of the C-type pathway. The 100-base pair left side sequence of ColE1 (colL) was subjected to systematic deletion using Bal31 exonucleolysis, showing that removal of 30 base pairs from its right end abolished extrusion by the C-type process. A cloned oligonucleotide of the same 30-base pair sequence was sufficient to confer C-type cruciform extrusion on an adjacent inverted repeat. An A + T-rich sequence from Drosophila was found to act like the ColE1 sequences. We have studied the effects of introducing sequences between the A + T-rich colL, and the inverted repeat on which it acts. A range of such fragments was found, from those which augment the effect of colL to those which block it completely. In general, it appears that the ability of a sequence to block the effect of colL depends on both the length and G + C content of the fragment. The sequences which are responsible for the extrusion by the C-type pathway are termed C-type inducing sequences, while sequences which are interposed between the inducing sequence and the inverted repeat, and which may either augment or attenuate the effect, but which cannot function as inducing sequences in isolation, are termed transmitting sequences. The results of these studies are most readily consistent with long range destabilization of DNA structure via telestability effects.     

Histone-histone interaction mediates chromatin unfolding at physiological ionic strength

High-resolution thermal denaturation data on chicken erythrocyte chromatin are reported over 4 orders of magnitude in NaCl concentration which includes the physiological region. A novel technique using critical-point polyacrylamide sols instead of ordinary solvents effectively stabilizes chromatin against precipitation at high salt concentrations. These sols are optically transparent from 260 to 320 nm and are thermally stable over the temperature ranges studied. At Na+ ion concentrations below 10 mM, the polyacrylamide slightly destabilizes chromatin at the nucleosome level, possibly through interactions of histones H1 and H5 with the carboxylic acid residues. At the same low salts, polyacrylamide stabilizes pure DNA against denaturation, presumably by mechanically stabilizing it against helix-distorting thermal fluctuations. In both cases, however, the polyacrylamide sols are entirely noninvasive at higher salts. Prominent low-temperature thermal transitions are observed in chromatin at and above 100 mM NaCl which evidently are associated with conformational changes in DNA. Our results are in accord with the idea that histone-histone interactions at physiological ionic strengths (approximately 100 mM Na+) may be comparable to histone-DNA interactions and hence may be sufficient to promote the destabilization of the DNA helix in chromatin under these conditions. The biological implications of this are discussed, and a possible model for the local decondensation of chromatin under physiological conditions is proposed.     

Physiologically relevant concentrations of NaCl and KCl increase DNA photocleavage by an N-substituted 9-aminomethylanthracene dye

This paper describes the synthesis of a new 9-aminomethylanthracene dye N-substituted with a pyridinylpolyamine side chain (4). The effects of NaCl and KCl on anthracene/DNA interactions were then studied, with the goal of simulating the conditions of high ionic strength that a DNA photosensitizer might encounter in the cell nucleus (~150 mM of NaCl and 260 mM of KCl). As exemplified by methylene blue (5), the expected effect of increasing ionic strength is to decrease DNA binding and photocleavage yields. In contrast, the addition of 150 mM of NaCl in combination with 260 mM of KCl to photocleavage reactions containing micromolar concentrations of 4 triggers the conversion of supercoiled, nicked, and linear forms of pUC19 plasmid into a highly degraded band of DNA fragments (350 nm hν, pH 7.0). Circular dichroism spectra point to a correlation between salt-induced unwinding of the DNA helix and the increase in DNA photocleavage yields. The results of circular dichroism, UV-vis absorption, fluorescence emission, thermal denaturation, and photocleavage inhibition experiments suggest that the combination of salts causes a change in the DNA binding mode of 4 from intercalation to an external interaction. This in turn leads to an increase in the anthracene-sensitized production of DNA-damaging reactive oxygen species.     

Long-range structural effects in supercoiled DNA: statistical thermodynamics reveals a correlation between calculated cooperative melting and contextual influence on cruciform extrusion

We have previously described [K. M. Sullivan and D. M. J. Lilley (1986) Cell 47, 817-827] a set of sequences, called C-type inducing sequences, which cause cruciform extrusion by adjacent inverted repeats to occur by an abnormal kinetic pathway involving a large denatured region of DNA. In this paper we apply statistical thermodynamic DNA helix melting theory to these sequences. We find a marked correlation between the ability of sequences to confer C-type cruciform character experimentally and their calculated propensity to undergo cooperative melting, and no exceptions have been found. The correlations are both qualitative and quantitative. Thus the ColE1 flanking sequences behave as single melting units, while the DNA of the S-type plasmid pIRbke8 exhibits no propensity to melt in the region of the bke cruciform. The results of the calculations are also fully consistent with the following experimental observations: 1. the ability of the isolated colL and colR fragments of the ColE1 flanking sequences, as well as the short sequence col30, to confer C-type character; 2. C-type induction by an A + T rich Drosophila sequence; 3. low-temperature cruciform extrusion by an (AT)34 sequence; 4. the effect of changing sequences at a site 90 base pairs (bp) removed from the inverted repeat; 5. the effects of systematic deletion of the colL sequence; and 6. the effects of insertion of various sequences in between the colL sequence and the xke inverted repeat. These studies show that telestability effects on thermal denaturation as predicted from equilibrium helix melting theory of linear DNA molecules may explain all the features that are revealed by studying the extrusion of cruciforms in circular DNA molecules subjected to superhelical stress.     

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na⁺. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.     

Partial purification and characterization of an endonuclease from spinach that cleaves ultraviolet light-damaged duplex DNA

An endonuclease that cleaves ultraviolet light (UV)-damaged, supercoiled plasmid DNA was partially purified from spinach leaves (Spinacia oleracea) by a series of column chromatography steps. Dialysis of the enzyme against EDTA resulted in a greater than 90% loss of activity which could be fully restored following the addition of Zn2+, suggesting that divalent cations are associated with the active enzyme. The spinach endonuclease cleaved duplex, UV-damaged, end-labelled DNA of defined sequence at positions of adenine in the presence of salt (KH2PO4 or NaCl) concentrations of 50 mM or higher. Cleavage of UV-irradiated DNA was dose-dependent and increased steadily within a fluence range of 50-10,000 J/m2. The UV damage requirement and adenine cleavage specificity could be eliminated with lower salt concentrations (0-25 mM), suggesting that the endonuclease recognizes and incises single-stranded DNA. The properties of this enzyme, which we have termed nuclease SP, suggest that it may mediate a role in DNA repair and/or recombination processes in spinach.     

Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

Interarm interaction of DNA cruciform forming at a short inverted repeat sequence

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100-200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Rhodococcus sp. RB1 grows in the presence of high nitrate and nitrite concentrations and assimilates nitrate in moderately saline environments

Rhodococcus sp. RB1 was able to thrive in media with up to 0.9 M NaCl or KCl and in the presence of high concentrations of nitrate (up to 0.9 M) and nitrite (up to 60 mM), but only under oxic conditions. An adaptation period was not required for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl. Nitrate assimilation was limited by the carbon supply, but nitrite was not accumulated in the culture medium, even at nitrate concentrations as high as 0.8 M, thus suggesting that nitrite reduction does not limit nitrate assimilation. The presence of NaCl or KCl did not affect nitrate or nitrite uptake, which were completely inhibited by ammonium or glutamine. Rhodococcus sp. RB1 nitrate reductase had an apparent molecular mass of 142 kDa and used NADH and reduced bromophenol blue or viologens as electron donors, independently of the presence of salt. The enzyme was associated with an NADH-diaphorase activity and was induced by nitrate and repressed by ammonium or glutamine, thus showing typical biochemical and regulatory properties of bacterial assimilatory NADH-nitrate reductases. The enzyme was active in vitro in the presence of 3 M NaCl or KCI, but the maximal activity was observed at 0.5 M salt. Addition of 2 M NaCl increased the optimal temperature of the enzyme from 12 to 32 degrees C, but the optimal pH (10.3) was unaffected.     

Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions.

In the presence of high concentrations of the nonspecific polymer polyethylene glycol (PEG), intermolecular cohesive-end ligation with the DNA ligase from Escherichia coli was stimulated by high salt concentrations: 200 mM NaCl or 300 mM KCl in 10% (w/v) PEG 6000 solutions, and 100-200 mM NaCl or 150-300 mM KCl in 15% PEG 6000 solutions. Intermolecular blunt-end ligation with this ligase was also stimulated at 100-150 mM NaCl or 150-250 mM KCl in 15% PEG 6000 solutions. The extent of such intermolecular ligation increased and the salt concentrations at which ligation was stimulated extended to lower concentrations when we raised the temperature from 10 to 37 degrees C.     

Winding of the DNA helix by divalent metal ions.

When supercoiled pBR322 DNA was relaxed at 0 or 22 degrees C by topoisomerase I in the presence of the divalent cations Ca2+, Mn2+ or Co2+, the resulting distributions of topoisomers observed at 22 degrees C had positive supercoils, up to an average delta Lk value of +8.6 (for Ca2+at 0 degrees C), corresponding to an overwinding of the helix by 0.7 degrees/bp. An increase of the divalent cation concentration in the reaction mixture above 50 mM completely reversed the effect. When such ions were present in agarose electrophoresis gels, they caused a relaxation of positively supercoiled DNA molecules, and thus allowed a separation of strongly positively supercoiled topoisomers. The effect of divalent cations on DNA adds a useful tool for the study of DNA topoisomers, for the generation as well as separation of positively supercoiled DNA molecules.     

The interaction of RNA polymerase and DNA Effects on the helix-coil transition and light scattering

To characterize the interactions of RNA polymerase with DNA, we have investigated the thermal transitions of poly[d(A-T] bound to RNA polymerase from Escherichia coli and the aggregation properties of the enzyme with DNA. The melting curve of the DNA-enzyme complex demonstrates a sharply lowered melting temperature for part of the DNA, whereas for another fraction the double helix is stabilized. This indicates that the DNA-binding site of RNA polymerase serves two functions: (1) to disrupt the double helix at one point, and (2) to maintain the duplex form at other points. The aggregation of DNA and RNA polymerase has been monitored by turbidity measurements, and conditions have been delineated under which aggregation is minimized. Holoenzyme added to double-stranded DNA or single-stranded DNA has little or no tendency to aggregate under most conditions. Core enzyme, on the other hand, aggregates extensively with double-stranded DNA, and only under conditions of low salt (10 mM KCl), without Mg²⁺, or at high salt (300 mM KCl), with or without Mg²⁺, can this aggregation be eliminated. Core enzyme also does not aggregate in the presence of single-stranded DNA. These aggregation properties are interpreted as evidence for more than one DNA-binding site on RNA polymerase.