A Quantitative Measure of DNA Curvature Enabling the Comparison of Predicted Structures

Abstract

A growing body of data indicates that the equilibrium structures of some DNA fragments are curved and that curvature is sequence-directed. We describe a quantitative measure of DNA curvature that can be used for evaluating and comparing current proposed models for the molecular basis of DNA curvature. We demonstrate that this measure, in conjunction with any given prediction model, enables both the comparison of experimental data to predictions and the scanning of nucleotide sequence databases for potential curved regions.     

Kinetics of site-site interactions in supercoiled DNA with bent sequences

A curved DNA segment is known to adopt a preferred end loop localization in superhelical (sc) DNA and thus may organize the overall conformation of the molecule. Through this process it influences the probability of site juxtaposition. We addressed the effect of a curvature on site-site interactions quantitatively by measuring the kinetics of cross-linking of two biotinylated positions in scDNA by streptavidin. The DNA was biotinylated at either symmetric or asymmetric positions with respect to a curved insert via triplex-forming oligonucleotides (TFOs) modified with biotin. We used a quench-flow device to mix the DNA with the protein and scanning force microscopy to quantify the reaction products. As a measure of the interaction probability, rate constants of cross-linking and local concentrations j(M) of one biotinylated site in the vicinity of the other were determined and compared to Monte Carlo simulations for corresponding DNAs. In good agreement with the simulations, a j(M) value of 1.74 microM between two sites 500bp apart was measured for an scDNA without curvature. When a curvature was centered between the sites, the interaction probability increased about twofold over the DNA without curvature, significantly less than expected from the simulations. However, the relative differences of the interaction probabilities due to varied biotin positions with respect to the curvature agreed quantitatively with the theory.     

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.     

An Escherichia coli protein that preferentially binds to sharply curved DNA

We attempted to find Escherichia coli proteins which preferentially bind to a curved DNA sequence even in the presence of an excess amount of a non-curved DNA sequence as a competitor, mainly by means of a DNA-binding gel retardation assay. Since the two sequences used had nearly the same nucleotide compositions, including consecutive dA5 stretches, we reasoned that this strategy would allow us to identify proteins which preferentially recognize an overall DNA curvature. We purified such a protein from E. coli. Its preferential binding to the curved DNA was found to be inhibited by distamycin, which removes the curvature from appropriate DNA sequences. The purified protein was identified as the E. coli nucleoid protein, H-NS.     

A non-curved chicken lysozyme 5'' matrix attachment site is 3'' followed by a strongly curved DNA sequence.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.     

Local structure of DNA toroids reveals curvature-dependent intermolecular forces

In viruses and cells, DNA is closely packed and tightly curved thanks to polyvalent cations inducing an effective attraction between its negatively charged filaments. Our understanding of this effective attraction remains very incomplete, partly because experimental data is limited to bulk measurements on large samples of mostly uncurved DNA helices. Here we use cryo electron microscopy to shed light on the interaction between highly curved helices. We find that the spacing between DNA helices in spermine-induced DNA toroidal condensates depends on their location within the torus, consistent with a mathematical model based on the competition between electrostatic interactions and the bending rigidity of DNA. We use our model to infer the characteristics of the interaction potential, and find that its equilibrium spacing strongly depends on the curvature of the filaments. In addition, the interaction is much softer than previously reported in bulk samples using different salt conditions. Beyond viruses and cells, our characterization of the interactions governing DNA-based dense structures could help develop robust designs in DNA nanotechnologies.     

Centromere parC of plasmid R1 is curved

The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.     

Relationship between codon usage and sequence-dependent curvature of genomes

Static DNA curvature distributions of full-sequenced genomes and large DNA contigs from different organisms were calculated. Very distinctive differences among histogram profiles coming from archaebacteria, eubacteria, and eukaryotes were observed. Eubacterial profiles were, on average, more curved than were archaeal and eukaryotic profiles. A comparative analysis between real and randomized DNA sequences revealed that eubacterial genomes presented, overall, higher curvature values than random sequences. An opposite portrait was exhibited by archaeal and eukaryotic genomes. They displayed a lower frequency of curved regions than their corresponding randomized sequences. The contributions of coding and intergenic regions to the curvature profile were also analyzed. Intergenic regions, on average, were found to be more curved than the overall genomic sequences, especially in prokaryotic organisms. Nevertheless, because of their small size with respect to coding regions, the contribution of intergenic sequences to the overall curvature profile tended to be minor. A clear relationship between codon usage and DNA curvature was demonstrated, and a proposal of the possible coevolution of both systems is discussed. Finally, we present a procedure to quantify the deviation of a curvature profile from randomness through a formal statistical analysis.     

Z-curve: a computer program calculating DNA helical axis coordinates for three-dimensional graphic presentation of curvature.

In order to predict curvature of DNA fragments, we previously developed a computer program for simply calculating a vectorial sum of all individual roll, tilt and twist wedge angles between the nearest base pairs for a given DNA fragment [Lee et al., (1991)]. Now, a new program, called Z-curve, was developed to calculate three-dimensional coordinates of the helical center of each base pair along the DNA, using helical axis deviations from B-form DNA by wedge angles. The output file of the new program was designed to become an input file for a graphics program, Insight II. Thus, we were able to obtain three-dimensional graphic presentations of DNA helical axis curvatures of any length. It visualized spatial details of the DNA curvature, where and how much it curves, and to which direction. It also allowed calculation of the three-dimensional distance between two ends of a DNA fragment, which could provide a measure of its curvature. Here, three DNA fragments, both curved and straight, were subjected to the Z-curve and Insight II programs. The results showed that their curvature details could be visualized to the level of the base pair, whether the DNA fragments contained an oligo(A) track or not. Their estimated curvatures were consistent with the experimental results of permutation gel mobility assay.     

Supercoiling-induced DNA bending

Local DNA bending is a critical factor for numerous DNA functions including recognition of DNA by sequence-specific regulatory binding proteins. Negative DNA supercoiling increases both local and global DNA dynamics, and this dynamic flexibility can facilitate the formation of DNA-protein complexes. We have recently shown that apexes of supercoiled DNA molecules are sites that can promote the formation of an alternative DNA structure, a cruciform, suggesting that these positions in supercoiled DNA are under additional stress and perhaps have a distorted DNA geometry. To test this hypothesis, we used atomic force microscopy to directly measure the curvature of apical positions in supercoiled DNA. The measurements were performed for an inherently curved sequence formed by phased A tracts and a region of mixed sequence DNA. For this, we used plasmids in which an inverted repeat and A tract were placed at precise locations relative to each other. Under specific conditions, the inverted repeat formed a cruciform that was used as a marker for the unambiguous identification of the A tract location. When the A tract and cruciform were placed diametrically opposite, this yielded predominantly nonbranched plectonemic molecules with an extruded cruciform and A tract localized in the terminal loops. For both the curved A tract and mixed sequence nonbent DNA, their localization to an apex increased the angle of bending compared to that expected for DNA unconstrained in solution. This is consistent with increased helical distortion at an apical bend.     

Relating independent measures of DNA curvature: electrophoretic anomaly and cyclization efficiency

Electrophoretic methods are often used to measure DNA curvature and protein-induced DNA bending. Though convenient and widely-applied, quantitative analyses are generally limited to assays for which empirical calibration standards have been developed. Alternatively, solution-based cyclization of short DNA duplexes allows analysis of DNA curvature and bending from first principles, but a detailed understanding of this assay is still lacking. In this work, we demonstrate that calibration with an independent electrophoretic assay of DNA curvature permits interpretation of cyclization assay results in a quantitatively meaningful way. We systematically measure intrinsic DNA curvature in short duplexes using a well-established empirical ligation ladder assay. We then compare the results to those obtained from the analysis of the distribution of circular products obtained in simple enzymatic cyclization assays of the same duplexes when polymerized. A strong correlation between DNA curvature estimates from these two assays is obtained for DNA fragments between 150-300 bp in length. We discuss how this result might be used to improve quantitative analysis of protein-mediated bending events evaluated by cyclization methods. Our results suggest that measurements of DNA curvature obtained under similar conditions, in solution and in an acrylamide gel matrix, can be compared directly. The ability to correlate results of these simple assays may prove convenient in monitoring DNA curvature and flexibility.     

Intrinsic curvature in the VP1 gene of SV40: comparison of solution and gel results

DNA restriction fragments that are stably curved are usually identified by polyacrylamide gel electrophoresis because curved fragments migrate more slowly than normal fragments containing the same number of basepairs. In free solution, curved DNA molecules can be identified by transient electric birefringence (TEB) because they exhibit rotational relaxation times that are faster than those of normal fragments of the same size. In this article, the results observed in free solution and in polyacrylamide gels are compared for a highly curved 199-basepair (bp) restriction fragment taken from the VP1 gene in Simian Virus 40 (SV40) and various sequence mutants and insertion derivatives. The TEB method of overlapping fragments was used to show that the 199-bp fragment has an apparent bend angle of 46 +/- 2 degrees centered at sequence position 1922 +/- 2 bp. Four unphased A- and T-tracts and a mixed A3T4-tract occur within a span of approximately 60 bp surrounding the apparent bend center; for brevity, this 60-bp sequence element is called a curvature module. Modifying any of the A- or T-tracts in the curvature module by site-directed mutagenesis decreases the curvature of the fragment; replacing all five A- and T-tracts by random-sequence DNA causes the 199-bp mutant to adopt a normal conformation, with normal electrophoretic mobilities and birefringence relaxation times. Hence, stable curvature in this region of the VP1 gene is due to the five unphased A- and T- tracts surrounding the apparent bend center. Discordant solution and gel results are observed when long inverted repeats are inserted within the curvature module. These insertion derivatives migrate anomalously slowly in polyacrylamide gels but have normal, highly flexible conformations in free solution. Discordant solution and gel results are not observed if the insert does not contain a long inverted repeat or if the long inverted repeat is added to the 199-bp fragment outside the curvature module. The results suggest that long inverted repeats can form hairpins or cruciforms when they are located within a region of the helix backbone that is intrinsically curved, leading to large mobility anomalies in polyacrylamide gels. Hairpin/cruciform formation is not observed in free solution, presumably because of rapid conformational exchange. Hence, DNA restriction fragments that migrate anomalously slowly in polyacrylamide gels are not necessarily stably curved in free solution.     

Dehydrating agents sharply reduce curvature in DNAs containing A tracts.

The structural basis of DNA curvature remains elusive, because models for curvature based on crystallographic structures of molecules containing A tracts do not agree with any of the models for sequence-directed curvature based on solution studies. Here we demonstrate that the difference is probably due to MPD (2-methyl-2,4-pentanediol), the dehydrating agent commonly used in crystallography. One characteristic signature of curved DNA molecules is that they run anomalously slowly on polyacrylamide gels, appearing to be larger than they actually are. The gel anomalies of three curved DNAs from trypanosome kinetoplast minicircles drop monotonically with increasing MPD concentration, indicating that MPD straightens molecules that are curved in aqueous solution. This is not due to some non-specific effect of MPD on poly(dA) or polypurine tracts, because control molecules containing dA70 and dG43 run normally over the full range of MPD concentrations. Circular dichroism spectra are not affected by MPD, ruling out a conformational change to a structure outside the B-DNA family. The effect is not due to MPD-induced changes in phasing of the curved sequences, because MPD has virtually no effect on the linking numbers of relaxed plasmids containing either curved sequences or dA70. At the concentrations of MPD used in X-ray crystallography, the curvature of DNAs containing A tracts is substantially lower than in solution, which probably explains the ongoing discrepancies between the crystallographic results and models based on solution studies.     

Structural modifications induced by the mtDBP-C protein in the replication origin of Xenopus laevis mitochondrial DNA

The structure of the non-coding region of Xenopus laevis mitochondrial DNA has been studied by electron microscopy analysis of DNA molecules end-labelled with streptavidin-ferritin. We have shown that the effect of a protein modifying the shape of the DNA double-helix can be studied and precisely located by this method. It was found that the non-coding region contains curved segments and that the mitochondrial protein mtDBP-C preferentially enhances the curvature of the promoters-replication origin region.