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1.  Susceptibility to Superhelically Driven DNA Duplex Destabilization: A Highly Conserved Property of Yeast Replication Origins 
Strand separation is obligatory for several DNA functions, including replication. However, local DNA properties such as A+T content or thermodynamic stability alone do not determine the susceptibility to this transition in vivo. Rather, superhelical stresses provide long-range coupling among the transition behaviors of all base pairs within a topologically constrained domain. We have developed methods to analyze superhelically induced duplex destabilization (SIDD) in genomic DNA that take into account both this long-range stress-induced coupling and sequence-dependent local thermodynamic stability. Here we apply this approach to examine the SIDD properties of 39 experimentally well-characterized autonomously replicating DNA sequences (ARS elements), which function as replication origins in the yeast Saccharomyces cerevisiae. We find that these ARS elements have a strikingly increased susceptibility to SIDD relative to their surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do their immediately surrounding sequences, making them more than 2,000 times easier to open. Statistical analysis shows that the probability of this strong an association between SIDD sites and ARS elements arising by chance is approximately 4 × 10−10. This local enhancement of the propensity to separate to single strands under superhelical stress has obvious implications for origin function. SIDD properties also could be used, in conjunction with other known origin attributes, to identify putative replication origins in yeast, and possibly in other metazoan genomes.
Synopsis
Several DNA functions require the two strands of the DNA duplex to transiently separate. Examples include the initiation of gene expression and of DNA replication. Here the authors examine the strand separation properties of the DNA duplex at autonomously replicating sequences (ARS elements), which are the potential replication origins in yeast.
In vivo, susceptibility to strand separation does not depend only on local DNA properties such as adenine plus thymine content or thermodynamic stability. Rather, stresses imposed on the DNA in vivo couple together the strand-opening behaviors of all base pairs that experience them. The authors use computational methods for analyzing stress-driven strand separation to examine the susceptibility to opening of 39 experimentally well-characterized ARS elements. They show that these ARS elements have strikingly increased susceptibilities to stress-induced separation relative to the surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do surrounding sequences, making them more than 2,000 times easier to open. This enhanced susceptibility to stress-driven strand separation has obvious implications for the mechanisms that begin the process of replication. This property is also shared by bacterial and viral replication start points, suggesting that it may be a general attribute of replication origins.
doi:10.1371/journal.pcbi.0010007
PMCID: PMC1183513  PMID: 16103908
2.  SIDDBASE: a database containing the stress-induced DNA duplex destabilization (SIDD) profiles of complete microbial genomes 
Nucleic Acids Research  2005;34(Database issue):D373-D378.
Prokaryotic genomic DNA is generally negatively supercoiled in vivo. Many regulatory processes, including the initiation of transcription, are known to depend on the superhelical state of the DNA substrate. The stresses induced within DNA by negative superhelicity can destabilize the DNA duplex at specific sites. Various experiments have either shown or suggested that stress-induced DNA duplex destabilization (SIDD) is involved in specific regulatory mechanisms governing a variety of biological processes. We have developed methods to evaluate the SIDD properties of DNA sequences, including complete chromosomes. This analysis predicts the locations where the duplex becomes destabilized under superhelical stress. Previous studies have shown that the SIDD-susceptible sites predicted in this way occur at rates much higher than expected at random in transcriptional regulatory regions, and much lower than expected in coding regions. Analysis of the SIDD profiles of 42 bacterial genomes chosen for their diversity confirms this pattern. Predictions of SIDD sites have been used to identify potential genomic regulatory regions, and suggest both possible regulatory mechanisms involving stress-induced destabilization and experimental tests of these mechanisms. Here we describe the SIDDBASE database which enables users to retrieve and visualize the results of SIDD analyses of completely sequenced prokaryotic and archaeal genomes, together with their annotations. SIDDBASE is available at .
doi:10.1093/nar/gkj007
PMCID: PMC1347370  PMID: 16381890
3.  Theoretical Analysis of Competing Conformational Transitions in Superhelical DNA 
PLoS Computational Biology  2012;8(4):e1002484.
We develop a statistical mechanical model to analyze the competitive behavior of transitions to multiple alternate conformations in a negatively supercoiled DNA molecule of kilobase length and specified base sequence. Since DNA superhelicity topologically couples together the transition behaviors of all base pairs, a unified model is required to analyze all the transitions to which the DNA sequence is susceptible. Here we present a first model of this type. Our numerical approach generalizes the strategy of previously developed algorithms, which studied superhelical transitions to a single alternate conformation. We apply our multi-state model to study the competition between strand separation and B-Z transitions in superhelical DNA. We show this competition to be highly sensitive to temperature and to the imposed level of supercoiling. Comparison of our results with experimental data shows that, when the energetics appropriate to the experimental conditions are used, the competition between these two transitions is accurately captured by our algorithm. We analyze the superhelical competition between B-Z transitions and denaturation around the c-myc oncogene, where both transitions are known to occur when this gene is transcribing. We apply our model to explore the correlation between stress-induced transitions and transcriptional activity in various organisms. In higher eukaryotes we find a strong enhancement of Z-forming regions immediately 5′ to their transcription start sites (TSS), and a depletion of strand separating sites in a broad region around the TSS. The opposite patterns occur around transcript end locations. We also show that susceptibility to each type of transition is different in eukaryotes and prokaryotes. By analyzing a set of untranscribed pseudogenes we show that the Z-susceptibility just downstream of the TSS is not preserved, suggesting it may be under selection pressure.
Author Summary
The stresses imposed on DNA within organisms can drive the molecule from its standard B-form double-helical structure into other conformations at susceptible sites within the sequence. We present a theoretical method to calculate this transition behavior due to stresses induced by supercoiling. We also develop a numerical algorithm that calculates the transformation probability of each base pair in a user-specified DNA sequence under stress. We apply this method to analyze the competition between transitions to strand separated and left-handed Z-form structures. We find that these two conformations are both competitive under physiological environmental conditions, and that this competition is especially sensitive to temperature. By comparing its results to experimental data we also show that the algorithm properly describes the competition between melting and Z-DNA formation. Analysis of large gene sets from various organisms shows a correlation between sites of stress-induced transitions and locations that are involved in regulating gene expression.
doi:10.1371/journal.pcbi.1002484
PMCID: PMC3343103  PMID: 22570598
4.  Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions. 
Nucleic Acids Research  1990;18(22):6621-6624.
We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.
Images
PMCID: PMC332619  PMID: 2251122
5.  Competitive superhelical transitions involving cruciform extrusion 
Nucleic Acids Research  2013;41(21):9610-9621.
A DNA molecule under negative superhelical stress becomes susceptible to transitions to alternate structures. The accessible alternate conformations depend on base sequence and compete for occupancy. We have developed a method to calculate equilibrium distributions among the states available to such systems, as well as their average thermodynamic properties. Here we extend this approach to include superhelical cruciform extrusion at both perfect and imperfect inverted repeat (IR) sequences. We find that short IRs do not extrude cruciforms, even in the absence of competition. But as the length of an IR increases, its extrusion can come to dominate both strand separation and B-Z transitions. Although many IRs are present in human genomic DNA, we find that extrusion-susceptible ones occur infrequently. Moreover, their avoidance of transcription start sites in eukaryotes suggests that cruciform formation is rarely involved in mechanisms of gene regulation. We examine a set of clinically important chromosomal translocation breakpoints that occur at long IRs, whose rearrangement has been proposed to be driven by cruciform extrusion. Our results show that the susceptibilities of these IRs to cruciform formation correspond closely with their observed translocation frequencies.
doi:10.1093/nar/gkt733
PMCID: PMC3834812  PMID: 23969416
6.  Promoter prediction and annotation of microbial genomes based on DNA sequence and structural responses to superhelical stress 
BMC Bioinformatics  2006;7:248.
Background
In our previous studies, we found that the sites in prokaryotic genomes which are most susceptible to duplex destabilization under the negative superhelical stresses that occur in vivo are statistically highly significantly associated with intergenic regions that are known or inferred to contain promoters. In this report we investigate how this structural property, either alone or together with other structural and sequence attributes, may be used to search prokaryotic genomes for promoters.
Results
We show that the propensity for stress-induced DNA duplex destabilization (SIDD) is closely associated with specific promoter regions. The extent of destabilization in promoter-containing regions is found to be bimodally distributed. When compared with DNA curvature, deformability, thermostability or sequence motif scores within the -10 region, SIDD is found to be the most informative DNA property regarding promoter locations in the E. coli K12 genome. SIDD properties alone perform better at detecting promoter regions than other programs trained on this genome. Because this approach has a very low false positive rate, it can be used to predict with high confidence the subset of promoters that are strongly destabilized. When SIDD properties are combined with -10 motif scores in a linear classification function, they predict promoter regions with better than 80% accuracy. When these methods were tested with promoter and non-promoter sequences from Bacillus subtilis, they achieved similar or higher accuracies. We also present a strictly SIDD-based predictor for annotating promoter sequences in complete microbial genomes.
Conclusion
In this report we show that the propensity to undergo stress-induced duplex destabilization (SIDD) is a distinctive structural attribute of many prokaryotic promoter sequences. We have developed methods to identify promoter sequences in prokaryotic genomes that use SIDD either as a sole predictor or in combination with other DNA structural and sequence properties. Although these methods cannot predict all the promoter-containing regions in a genome, they do find large sets of potential regions that have high probabilities of being true positives. This approach could be especially valuable for annotating those genomes about which there is limited experimental data.
doi:10.1186/1471-2105-7-248
PMCID: PMC1468432  PMID: 16677393
7.  Superhelical Destabilization in Regulatory Regions of Stress Response Genes  
PLoS Computational Biology  2008;4(1):e17.
Stress-induced DNA duplex destabilization (SIDD) analysis exploits the known structural and energetic properties of DNA to predict sites that are susceptible to strand separation under negative superhelical stress. When this approach was used to calculate the SIDD profile of the entire Escherichia coli K12 genome, it was found that strongly destabilized sites occur preferentially in intergenic regions that are either known or inferred to contain promoters, but rarely occur in coding regions. Here, we investigate whether the genes grouped in different functional categories have characteristic SIDD properties in their upstream flanks. We report that strong SIDD sites in the E. coli K12 genome are statistically significantly overrepresented in the upstream regions of genes encoding transcriptional regulators. In particular, the upstream regions of genes that directly respond to physiological and environmental stimuli are more destabilized than are those regions of genes that are not involved in these responses. Moreover, if a pathway is controlled by a transcriptional regulator whose gene has a destabilized 5′ flank, then the genes (operons) in that pathway also usually contain strongly destabilized SIDD sites in their 5′ flanks. We observe this statistically significant association of SIDD sites with upstream regions of genes functioning in transcription in 38 of 43 genomes of free-living bacteria, but in only four of 18 genomes of endosymbionts or obligate parasitic bacteria. These results suggest that strong SIDD sites 5′ to participating genes may be involved in transcriptional responses to environmental changes, which are known to transiently alter superhelicity. We propose that these SIDD sites are active and necessary participants in superhelically mediated regulatory mechanisms governing changes in the global pattern of gene expression in prokaryotes in response to physiological or environmental changes.
Author Summary
DNA in vivo experiences regulated amounts of untwisting stress. If sufficiently large, these stresses can destabilize the double helix at specific locations. These sites then become favored locations for strand separations. Gene expression and DNA replication, the two major jobs of DNA, both require the strands of the duplex to be separated. Thus, events that affect the ease of strand separation can regulate the initiation of these processes. Stress-induced DNA duplex destabilization (SIDD) has been implicated in mechanisms regulating several biological processes, including the initiation of gene expression and replication. We have developed computational methods that accurately predict the locations and extents of destabilization within genomic DNA sequences that occur in response to specified stress levels. Here, we report that the easily destabilized sites we find in the Escherichia coli K12 genome are statistically significantly overrepresented in the upstream regions of genes encoding proteins that regulate transcription. In particular, the regions upstream of genes that directly respond to physiological and environmental stimuli are more destabilized than are those regions of genes that are not involved in these responses. These results suggest that strong SIDD sites upstream of participating genes may be involved in transcriptional responses to environmental changes.
doi:10.1371/journal.pcbi.0040017
PMCID: PMC2211533  PMID: 18208321
8.  Superhelical Destabilization in Regulatory Regions of Stress Response Genes  
PLoS Computational Biology  2008;4(1):e17.
Stress-induced DNA duplex destabilization (SIDD) analysis exploits the known structural and energetic properties of DNA to predict sites that are susceptible to strand separation under negative superhelical stress. When this approach was used to calculate the SIDD profile of the entire Escherichia coli K12 genome, it was found that strongly destabilized sites occur preferentially in intergenic regions that are either known or inferred to contain promoters, but rarely occur in coding regions. Here, we investigate whether the genes grouped in different functional categories have characteristic SIDD properties in their upstream flanks. We report that strong SIDD sites in the E. coli K12 genome are statistically significantly overrepresented in the upstream regions of genes encoding transcriptional regulators. In particular, the upstream regions of genes that directly respond to physiological and environmental stimuli are more destabilized than are those regions of genes that are not involved in these responses. Moreover, if a pathway is controlled by a transcriptional regulator whose gene has a destabilized 5′ flank, then the genes (operons) in that pathway also usually contain strongly destabilized SIDD sites in their 5′ flanks. We observe this statistically significant association of SIDD sites with upstream regions of genes functioning in transcription in 38 of 43 genomes of free-living bacteria, but in only four of 18 genomes of endosymbionts or obligate parasitic bacteria. These results suggest that strong SIDD sites 5′ to participating genes may be involved in transcriptional responses to environmental changes, which are known to transiently alter superhelicity. We propose that these SIDD sites are active and necessary participants in superhelically mediated regulatory mechanisms governing changes in the global pattern of gene expression in prokaryotes in response to physiological or environmental changes.
Author Summary
DNA in vivo experiences regulated amounts of untwisting stress. If sufficiently large, these stresses can destabilize the double helix at specific locations. These sites then become favored locations for strand separations. Gene expression and DNA replication, the two major jobs of DNA, both require the strands of the duplex to be separated. Thus, events that affect the ease of strand separation can regulate the initiation of these processes. Stress-induced DNA duplex destabilization (SIDD) has been implicated in mechanisms regulating several biological processes, including the initiation of gene expression and replication. We have developed computational methods that accurately predict the locations and extents of destabilization within genomic DNA sequences that occur in response to specified stress levels. Here, we report that the easily destabilized sites we find in the Escherichia coli K12 genome are statistically significantly overrepresented in the upstream regions of genes encoding proteins that regulate transcription. In particular, the regions upstream of genes that directly respond to physiological and environmental stimuli are more destabilized than are those regions of genes that are not involved in these responses. These results suggest that strong SIDD sites upstream of participating genes may be involved in transcriptional responses to environmental changes.
doi:10.1371/journal.pcbi.0040017
PMCID: PMC2211533  PMID: 18208321
9.  The influence of tertiary structural restraints on conformational transitions in superhelical DNA. 
Nucleic Acids Research  1987;15(23):9985-9995.
This paper examines theoretically the effects that restraints on the tertiary structure of a superhelical DNA domain exert on the energetics of linking and the onset of conformational transitions. The most important tertiary constraint arises from the nucleosomal winding of genomic DNA in vivo. Conformational transitions are shown to occur at equilibrium at less extreme superhelicities in DNA whose tertiary structure is restrained than in unrestrained molecules where the residual linking difference alpha res (that part of the superhelical deformation which is not absorbed by transitions) may be freely partitioned between twisting and bending. In the extreme case of a rigidly held tertiary structure, this analysis predicts that the B-Z transition will occur at roughly half the superhelix density needed to drive the same transition in solution, other factors remaining fixed. This suggests that superhelical transitions may occur at more moderate superhelical deformations in vivo than in solution. The influence on transition behavior of the tertiary structural restraints imposed by gel conditions also are discussed.
PMCID: PMC306545  PMID: 3320960
10.  Superhelical Duplex Destabilization and the Recombination Position Effect 
PLoS ONE  2011;6(6):e20798.
The susceptibility to recombination of a plasmid inserted into a chromosome varies with its genomic position. This recombination position effect is known to correlate with the average G+C content of the flanking sequences. Here we propose that this effect could be mediated by changes in the susceptibility to superhelical duplex destabilization that would occur. We use standard nonparametric statistical tests, regression analysis and principal component analysis to identify statistically significant differences in the destabilization profiles calculated for the plasmid in different contexts, and correlate the results with their measured recombination rates. We show that the flanking sequences significantly affect the free energy of denaturation at specific sites interior to the plasmid. These changes correlate well with experimentally measured variations of the recombination rates within the plasmid. This correlation of recombination rate with superhelical destabilization properties of the inserted plasmid DNA is stronger than that with average G+C content of the flanking sequences. This model suggests a possible mechanism by which flanking sequence base composition, which is not itself a context-dependent attribute, can affect recombination rates at positions within the plasmid.
doi:10.1371/journal.pone.0020798
PMCID: PMC3111454  PMID: 21695263
11.  DNA Conformational Transitions Induced by Supercoiling Control Transcription in Chromatin 
Regulation of transcription in eukaryotes is considered in the light of recent findings demonstrating the presence of negative and positive superhelical tension in chromatin. This tension induces conformational transitions in DNA duplex. Particularly, the transition into A-form renders DNA accessible and waylaying for initiation of transcription producing RNA molecules long known to belong to the A-conformation. Competition between conformational transitions in various DNA sequences for the energy of elastic spring opens a possibility for understanding of fine tuning of transcription at a distance.
doi:10.4137/GRSB.S13756
PMCID: PMC3956857  PMID: 24653646
DNA conformation; nucleosomes; transcription; RNA-polymerase; topoisomerase II
12.  Fluctuations in superhelical DNA. 
Nucleic Acids Research  1979;6(3):967-982.
The effect of superhelicity on the base-pair opening probability and on the probability of occurrence of cruciform states in palindromic regions is theoretically treated. The calculations show that below the superhelix density value of -sigma=0.05 superhelicity does not appreciably affect the characteristics of DNA secondary structure fluctuations. In the range of physiological superhelix densities sigma (-sigma=0.05-0.09) the base-pair opening probability markedly increases. However, within this range of sigma the base-pairs are opened only transiently and permanently open regions are not formed. Permanently opened regions appear at higher negative superhelix densities (-sigma greater than 0.10). At the values of -sigma higher than 0.06 a cruciform structure in the palindromic region centred in position 3965 proves to be the most probable fluctuational disturbance in the 0x174 duplex DNA. Different experimental approaches used for probing the fluctuations in superhelical DNA have been analysed. The results suggest that most direct quantitative information can be derived from data on the nicking of closed DNA by single strand-specific endonucleases. Such data (Wang, 1974) accord with the results of theoretical calculations. Calculations show that, due to base-pair opening, the total free energy of superhelical DNA should depend parabolically on sigma only up to some critical value of sigma=sigmac. If negative superhelicity exceeds this critical value, which under physiological conditions proves to be -sigma=0.085, the free energy should increase linearly with -sigma. The biological role of supercoiling is discussed in the light of obtained results.
PMCID: PMC327745  PMID: 155809
13.  Mapping the phase diagram of the writhe of DNA nanocircles using atomistic molecular dynamics simulations 
Nucleic Acids Research  2007;36(1):21-29.
We have investigated the effects of duplex length, sequence, salt concentration and superhelical density on the conformation of DNA nanocircles containing up to 178 base pairs using atomistic molecular dynamics simulation. These calculations reveal that the partitioning of twist and writhe is governed by a delicate balance of competing energetic terms. We have identified conditions which favour circular, positively or negatively writhed and denatured DNA conformations. Our simulations show that AT-rich DNA is more prone to denaturation when subjected to torsional stress than the corresponding GC containing circles. In contrast to the behaviour expected for a simple elastic rod, there is a distinct asymmetry in the behaviour of over and under-wound DNA nanocircles. The most biologically relevant negatively writhed state is more elusive than the corresponding positively writhed conformation, and is only observed for larger circles under conditions of high electrostatic screening. The simulation results have been summarised by plotting a phase diagram describing the various conformational states of nanocircles over the range of circle sizes and experimental conditions explored during the study. The changes in DNA structure that accompany supercoiling suggest a number of mechanisms whereby changes in DNA topology in vivo might be used to influence gene expression.
doi:10.1093/nar/gkm891
PMCID: PMC2248748  PMID: 17984075
14.  Studies on the interaction of H1 histone with superhelical DNA: characterization of the recognition and binding regions of H1 histones. 
Nucleic Acids Research  1976;3(10):2531-2547.
The very lysine rich histone, H1, isolated from a variety of sources interacts preferentially with superhelical DNA compared to relaxed DNA duplexes. The nature of this specific interaction has been investigated by studying the ability of various purified fragments of H1 histone from calf thymus to recognize and bind superhelical DNA. The data suggest that the globular region of the H1 histone molecule (amino acid residues 72-106) is involved in the recognition of superhelical DNA. Thus, the H1 histone carboxy-terminal fragment, 72-212, resembles native H1 histone both quantitatively and qualitatively in its ability to discriminate between and bind to superhelical and relaxed DNA while the H1 histone carboxy-terminal fragment, residues 106-212, has lost this specificity, binding superhelical and relaxed DNA equally well. Furthermore, under conditions in which the globular region of the intact H1 histone has been unfolded, the molecule loses its ability to discriminate between superhelical and relaxed DNA, and binds both forms of DNA equally.
Images
PMCID: PMC343111  PMID: 186761
15.  The effect of zinc on the secondary structure of d(GA.TC)n DNA sequences of different length: a model for the formation *H-DNA. 
Nucleic Acids Research  1993;21(11):2557-2562.
Alternating d(GA.TC)n DNA sequences are known to undergo transition to *H-DNA in the presence of zinc. Here, the effect of zinc on the secondary DNA structure of d(GA.TC)n sequences of different length (n = 5, 8, 10 and 19) was determined. Short d(GA.TC)n sequences form *H-DNA with a higher difficulty than longer ones. At bacterial negative superhelical density (- sigma = 0.05), zinc still induces transition to the *H-DNA conformation at a d(GA.TC)10 sequence but shorter sequences do not form *H-DNA. Transition to *H-DNA at a d(GA.TC)8 sequence is observed under conditions which destabilize the DNA double helix such as high negative supercoiling or low ionic strength. Our results indicate that a first step in the transition to *H-DNA is the formation of a denaturation bubble at the centre of the repeated DNA sequence, suggesting that the primary role of zinc is to induce a local denaturation of the DNA double helix. Subsequently, zinc might also participate in the stabilization of the altered DNA conformation through its direct interaction with the bases. Based on these results a model for the formation of *H-DNA is proposed.
Images
PMCID: PMC309581  PMID: 8332452
16.  The B-A transition in superhelical DNA. 
Nucleic Acids Research  1990;18(4):759-761.
Relaxation of a DNA superhelical stress due to the B to A transition induced by trifluoroethanol has been studied by assessing the change of DNA orientation in a flow gradient. Using DNAs of different superhelical densities, a decrease in the winding angle during the B----A shift of DNA was found to be 1.5 degrees per base pair in solution. Accepting the winding angle for B-DNA in solution to be 34.1 degrees, that for A-DNA must have a value of 32.6 degrees which agrees with the X-ray data for A-DNA in the condensed state. The date obtained within the B-A transition interval make it possible to conclude that there is an increase in winding at each B/A junction, which is about 5 degrees per one junction.
PMCID: PMC330324  PMID: 2156228
17.  Analysis of stress-induced duplex destabilization (SIDD) properties of replication origins, genes and intergenes in the fission yeast, Schizosaccharomyces pombe 
BMC Research Notes  2012;5:643.
Background
Replication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD) for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe.
Results
We have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs) and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD’s role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region.
Conclusion
SIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.
doi:10.1186/1756-0500-5-643
PMCID: PMC3533806  PMID: 23163955
Replication origins; ARS elements; S. pombe; SIDD
18.  Early melting of supercoiled DNA. 
Nucleic Acids Research  1988;16(8):3269-3281.
Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.
Images
PMCID: PMC336493  PMID: 3375056
19.  Promoter prediction in E. coli based on SIDD profiles and Artificial Neural Networks 
BMC Bioinformatics  2010;11(Suppl 6):S17.
Background
One of the major challenges in biology is the correct identification of promoter regions. Computational methods based on motif searching have been the traditional approach taken. Recent studies have shown that DNA structural properties, such as curvature, stacking energy, and stress-induced duplex destabilization (SIDD) are useful in promoter prediction, as well. In this paper, the currently used SIDD energy threshold method is compared to the proposed artificial neural network (ANN) approach for finding promoters based on SIDD profile data.
Results
When compared to the SIDD threshold prediction method, artificial neural networks showed noticeable improvements for precision, recall, and F-score over a range of values. The maximal F-score for the ANN classifier was 62.3 and 56.8 for the threshold-based classifier.
Conclusions
Artificial neural networks were used to predict promoters based on SIDD profile data. Results using this technique were an improvement over the previous SIDD threshold approach. Over a wide range of precision-recall values, artificial neural networks were more capable of identifying distinctive characteristics of promoter regions than threshold based methods.
doi:10.1186/1471-2105-11-S6-S17
PMCID: PMC3026364  PMID: 20946600
20.  Conformational DNA transition in the in vitro torsionally strained chicken beta-globin 5' region. 
Nucleic Acids Research  1986;14(18):7143-7158.
A sequence of 86 bp within the 5' region of the adult chicken beta-globin gene was found to undergo a DNA conformational transition at elevated levels of negative superhelical stress (- sigma = 0.068). In vitro chemical DNA modification studies which detect purine hyperreactivity (HR) to the alkylating agent diethyl pyrocarbonate (DEP) have identified this 86 bp long DEP-HR element. The DEP-HR element is composed of small, tandem segments with imperfect purine-pyrimidine alternations. Methylation of cytosines within GCGC sequences of the DEP-HR element facilitates this structural change. The binding of a monoclonal anti-Z-DNA antibody to the element has been revealed by chemical footprinting with DEP. These data suggest that the DEP-HR sequence can undergo a conformational transition to Z-DNA. It is unknown whether the conformational flexibility observed here occurs in vivo.
Images
PMCID: PMC311742  PMID: 3763402
21.  The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression 
Journal of medicinal chemistry  2009;52(9):2863-2874.
The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc.
doi:10.1021/jm900055s
PMCID: PMC2757002  PMID: 19385599
22.  Locating Interrupted Hydrogen Bonding in the Secondary Structure of PM2 Circular DNA by Comparative Denaturation Mapping 
Journal of Virology  1974;13(6):1176-1185.
Previous studies with HCHO have revealed a reaction with superhelical DNA that strongly suggests that this DNA consists of small regions of interrupted secondary structure. To map these sites in PM2 DNA, the following set of experiments was performed using electron microscopy. (i) A denaturation map of nicked form II was obtained using Inman's alkaline-HCHO conditions. (ii) The superhelical form I was reacted with HCHO at 30 C until equilibrium was achieved at the interrupted sites (3.6% reactivity). The excess HCHO was removed rapidly and X-ray treatment was employed to nick these prereacted molecules. These form II molecules containing HCHO (form II HCHO) were also subjected to denaturation mapping. It would be expected that the HCHO-unpaired regions would serve as induction sites for the propagation of melting. Hence, depending on the location of the induction sites; we would anticipate either the creation of new regions of melting or a normal denaturation map shifted to lower pH values. Comparison of the development of progressive denaturation of form II and form II HCHO reveals that the latter is the case. The denaturation maps of form II are highly organized patterns of adenine-thymine (AT)-rich regions, with a total of five regions at extreme pH conditions. There are six highly organized regions for form II HCHO, i.e., smaller adjacent loops, at low denaturation conditions where no denaturation is seen for form II. These coalesce into the pattern for form II containing four of five A-T-rich regions observed for form II. Hence we conclude that the regions of altered hydrogen bonding in superhelical PM2 DNA are four to six in number and they map in the A-T-rich regions of the DNA.
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PMCID: PMC355436  PMID: 4833606
23.  Virus with a Multipartite Superhelical DNA Genome from the Ichneumonid Parasitoid Campoletis sonorensis 
Journal of Virology  1982;43(3):859-870.
Virus was isolated from the lumen of the calyx region of ovaries in the parasitoid wasp Campoletis sonorensis (Hymenoptera: Ichneumonidae), and the nature of the viral DNA was analyzed. DNA purified from a homogeneous band of virus contained double-stranded superhelical molecules which were polydisperse in molecular weight. At least 25 different covalently closed circles were present, ranging in molecular weight from 4.0 × 106 to 13.6 × 106. The virus DNA was analyzed with restriction enzymes, and the nature of the genetic complexity was evaluated by Southern blot hybridization of native superhelical and relaxed circular virus DNA and of SalI- and HindIII-digested DNA. The data suggest that most of the variously sized covalently closed DNAs were composed primarily of nonhomologous sequences. The different size classes of covalently closed viral DNAs did not appear to exist in equimolar concentrations. However, there was no evidence from observation of virus particles in the electron microscope or from virus fractionation experiments that a mixture of viruses was present in the calyx fluid. The results from this study suggest' that the virus isolated from C. sonorensis, like those isolated from other endoparasitic hymenoptera, may belong to a new class of DNA viruses in which the genome is multipartite, with each DNA existing as a superhelical molecule.
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PMCID: PMC256196  PMID: 16789230
24.  Early melting of supercoiled DNA topoisomers observed by TGGE 
Nucleic Acids Research  2000;28(11):e51.
We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (σ). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topoisomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with σ < –0.05 is very significant because these temperatures for early melting are below physiological temperatures.
PMCID: PMC102636  PMID: 10871350
25.  d(TG)n.d(CA)n sequences upstream of the rat prolactin gene form Z-DNA and inhibit gene transcription. 
Nucleic Acids Research  1990;18(6):1595-1601.
Two alternating purine-pyrimidine sequences of the d(TG)n.d(CA)n-type (170bp and 60 bp in length) lie upstream of the rat prolactin (rPRL) gene. Conformational studies of plasmids containing these sequences indicate that both form left-handed (Z) DNA, with transitions initiating at superhelical densities of -0.041 and -0.044 respectively. These alternating purine-pyrimidine (APP) sequences are hypersensitive to cleavage with S1 nuclease both at the boundaries and within these APP repeats, where there is a loss in APP alternation. We have investigated the function of one of these Z-DNA sequences in the regulation of rPRL transcription, by linking regions of the 5' flanking sequence of the rPRL gene to a reporter gene encoding chloramphenicol acetyltransferase (CAT), and transferring these plasmids into GH3 pituitary tumour cell lines. The major conclusion from these studies is that the 170bp repeat exerts a negative effect on the transcription of the rPRL gene, and also down-regulates the expression of the fusion gene pRSVcat when cloned 50bp upstream of the Rous sarcoma virus promoter. However, despite its proximity to an estrogen response element in prolactin, this sequence does not affect the responsiveness of the rPRL gene to estrogen.
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PMCID: PMC330531  PMID: 2158081

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