DNA superhelicity.

Closing each strand of a DNA duplex upon itself fixes its linking number L. This topological condition couples together the secondary and tertiary structures of the resulting ccDNA topoisomer, a constraint that is not present in otherwise identical nicked or linear DNAs. Fixing L has a range of structural, energetic and functional consequences. Here we consider how L having different integer values (that is, different superhelicities) affects ccDNA molecules. The approaches used are primarily theoretical, and are developed from a historical perspective. In brief, processes that either relax or increase superhelicity, or repartition what is there, may either release or require free energy. The energies involved can be substantial, sufficient to influence many events, directly or indirectly. Here two examples are developed. The changes of unconstrained superhelicity that occur during nucleosome attachment and release are examined. And a simple theoretical model of superhelically driven DNA structural transitions is described that calculates equilibrium distributions for populations of identical topoisomers. This model is used to examine how these distributions change with superhelicity and other factors, and applied to analyze several situations of biological interest.

Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation.

Many cellular processes occur out of equilibrium. This includes site-specific unwinding in supercoiled DNA, which may play an important role in gene regulation. Here, we use the Convex Lens-induced Confinement (CLiC) single-molecule microscopy platform to study these processes with high-throughput and without artificial constraints on molecular structures or interactions. We use two model DNA plasmid systems, pFLIP-FUSE and pUC19, to study the dynamics of supercoiling-induced secondary structural transitions after perturbations away from equilibrium. We find that structural transitions can be slow, leading to long-lived structural states whose kinetics depend on the duration and direction of perturbation. Our findings highlight the importance of out-of-equilibrium studies when characterizing the complex structural dynamics of DNA and understanding the mechanisms of gene regulation.

Interplay between DNA sequence and negative superhelicity drives R-loop structures.

R-loops are abundant three-stranded nucleic-acid structures that form in cis during transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms-junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

Emerging roles for R-loop structures in the management of topological stress.

R-loop structures are a prevalent class of alternative non-B DNA structures that form during transcription upon invasion of the DNA template by the nascent RNA. R-loops form universally in the genomes of organisms ranging from bacteriophages, bacteria, and yeasts to plants and animals, including mammals. A growing body of work has linked these structures to both physiological and pathological processes, in particular to genome instability. The rising interest in R-loops is placing new emphasis on understanding the fundamental physicochemical forces driving their formation and stability. Pioneering work in Escherichia coli revealed that DNA topology, in particular negative DNA superhelicity, plays a key role in driving R-loops. A clear role for DNA sequence was later uncovered. Here, we review and synthesize available evidence on the roles of DNA sequence and DNA topology in controlling R-loop formation and stability. Factoring in recent developments in R-loop modeling and single-molecule profiling, we propose a coherent model accounting for the interplay between DNA sequence and DNA topology in driving R-loop structure formation. This model reveals R-loops in a new light as powerful and reversible topological stress relievers, an insight that significantly expands the repertoire of R-loops' potential biological roles under both normal and aberrant conditions.

Single-molecule visualization of the effects of ionic strength and crowding on structure-mediated interactions in supercoiled DNA molecules.

DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe-plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe-plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNA's proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe-plasmid interactions once the site is unwound.

Visualizing structure-mediated interactions in supercoiled DNA molecules.

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid's structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

Long-range correlations in the mechanics of small DNA circles under topological stress revealed by multi-scale simulation.

It is well established that gene regulation can be achieved through activator and repressor proteins that bind to DNA and switch particular genes on or off, and that complex metabolic networks determine the levels of transcription of a given gene at a given time. Using three complementary computational techniques to study the sequence-dependence of DNA denaturation within DNA minicircles, we have observed that whenever the ends of the DNA are constrained, information can be transferred over long distances directly by the transmission of mechanical stress through the DNA itself, without any requirement for external signalling factors. Our models combine atomistic molecular dynamics (MD) with coarse-grained simulations and statistical mechanical calculations to span three distinct spatial resolutions and timescale regimes. While they give a consensus view of the non-locality of sequence-dependent denaturation in highly bent and supercoiled DNA loops, each also reveals a unique aspect of long-range informational transfer that occurs as a result of restraining the DNA within the closed loop of the minicircles.

SIST: stress-induced structural transitions in superhelical DNA.

SUMMARY: Supercoiling imposes stress on a DNA molecule that can drive susceptible sequences into alternative non-B form structures. This phenomenon occurs frequently in vivo and has been implicated in biological processes, such as replication, transcription, recombination and translocation. SIST is a software package that analyzes sequence-dependent structural transitions in kilobase length superhelical DNA molecules. The numerical algorithms in SIST are based on a statistical mechanical model that calculates the equilibrium probability of transition for each base pair in the domain. They are extensions of the original stress-induced duplex destabilization (SIDD) method, which analyzes stress-driven DNA strand separation. SIST also includes algorithms to analyze B-Z transitions and cruciform extrusion. The SIST pipeline has an option to use the DZCBtrans algorithm, which analyzes the competition among these three transitions within a superhelical domain. AVAILABILITY AND IMPLEMENTATION: The package and additional documentation are freely available at https://bitbucket.org/benhamlab/sist_codes. CONTACT: dzhabinskaya@ucdavis.edu.

Potential non-B DNA regions in the human genome are associated with higher rates of nucleotide mutation and expression variation.

While individual non-B DNA structures have been shown to impact gene expression, their broad regulatory role remains elusive. We utilized genomic variants and expression quantitative trait loci (eQTL) data to analyze genome-wide variation propensities of potential non-B DNA regions and their relation to gene expression. Independent of genomic location, these regions were enriched in nucleotide variants. Our results are consistent with previously observed mutagenic properties of these regions and counter a previous study concluding that G-quadruplex regions have a reduced frequency of variants. While such mutagenicity might undermine functionality of these elements, we identified in potential non-B DNA regions a signature of negative selection. Yet, we found a depletion of eQTL-associated variants in potential non-B DNA regions, opposite to what might be expected from their proposed regulatory role. However, we also observed that genes downstream of potential non-B DNA regions showed higher expression variation between individuals. This coupling between mutagenicity and tolerance for expression variability of downstream genes may be a result of evolutionary adaptation, which allows reconciling mutagenicity of non-B DNA structures with their location in functionally important regions and their potential regulatory role.

Competitive superhelical transitions involving cruciform extrusion.

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.

The genome-wide distribution of non-B DNA motifs is shaped by operon structure and suggests the transcriptional importance of non-B DNA structures in Escherichia coli.

Although the right-handed double helical B-form DNA is most common under physiological conditions, DNA is dynamic and can adopt a number of alternative structures, such as the four-stranded G-quadruplex, left-handed Z-DNA, cruciform and others. Active transcription necessitates strand separation and can induce such non-canonical forms at susceptible genomic sequences. Therefore, it has been speculated that these non-B DNA motifs can play regulatory roles in gene transcription. Such conjecture has been supported in higher eukaryotes by direct studies of several individual genes, as well as a number of large-scale analyses. However, the role of non-B DNA structures in many lower organisms, in particular proteobacteria, remains poorly understood and incompletely documented. In this study, we performed the first comprehensive study of the occurrence of B DNA-non-B DNA transition-susceptible sites (non-B DNA motifs) within the context of the operon structure of the Escherichia coli genome. We compared the distributions of non-B DNA motifs in the regulatory regions of operons with those from internal regions. We found an enrichment of some non-B DNA motifs in regulatory regions, and we show that this enrichment cannot be simply explained by base composition bias in these regions. We also showed that the distribution of several non-B DNA motifs within intergenic regions separating divergently oriented operons differs from the distribution found between convergent ones. In particular, we found a strong enrichment of cruciforms in the termination region of operons; this enrichment was observed for operons with Rho-dependent, as well as Rho-independent terminators. Finally, a preference for some non-B DNA motifs was observed near transcription factor-binding sites. Overall, the conspicuous enrichment of transition-susceptible sites in these specific regulatory regions suggests that non-B DNA structures may have roles in the transcriptional regulation of specific operons within the E. coli genome.

Theoretical analysis of competing conformational transitions in superhelical DNA.

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.

Theoretical analysis of the stress induced B-Z transition in superhelical DNA.

We present a method to calculate the propensities of regions within a DNA molecule to transition from B-form to Z-form under negative superhelical stresses. We use statistical mechanics to analyze the competition that occurs among all susceptible Z-forming regions at thermodynamic equilibrium in a superhelically stressed DNA of specified sequence. This method, which we call SIBZ, is similar to the SIDD algorithm that was previously developed to analyze superhelical duplex destabilization. A state of the system is determined by assigning to each base pair either the B- or the Z-conformation, accounting for the dinucleotide repeat unit of Z-DNA. The free energy of a state is comprised of the nucleation energy, the sequence-dependent B-Z transition energy, and the energy associated with the residual superhelicity remaining after the change of twist due to transition. Using this information, SIBZ calculates the equilibrium B-Z transition probability of each base pair in the sequence. This can be done at any physiologically reasonable level of negative superhelicity. We use SIBZ to analyze a variety of representative genomic DNA sequences. We show that the dominant Z-DNA forming regions in a sequence can compete in highly complex ways as the superhelicity level changes. Despite having no tunable parameters, the predictions of SIBZ agree precisely with experimental results, both for the onset of transition in plasmids containing introduced Z-forming sequences and for the locations of Z-forming regions in genomic sequences. We calculate the transition profiles of 5 kb regions taken from each of 12,841 mouse genes and centered on the transcription start site (TSS). We find a substantial increase in the frequency of Z-forming regions immediately upstream from the TSS. The approach developed here has the potential to illuminate the occurrence of Z-form regions in vivo, and the possible roles this transition may play in biological processes.

DNA stress and strain, in silico, in vitro and in vivo.

A vast literature has explored the genetic interactions among the cellular components regulating gene expression in many organisms. Early on, in the absence of any biochemical definition, regulatory modules were conceived using the strict formalism of genetics to designate the modifiers of phenotype as either cis- or trans-acting depending on whether the relevant genes were embedded in the same or separate DNA molecules. This formalism distilled gene regulation down to its essence in much the same way that consideration of an ideal gas reveals essential thermodynamic and kinetic principles. Yet just as the anomalous behavior of materials may thwart an engineer who ignores their non-ideal properties, schemes to control and manipulate the genetic and epigenetic programs of cells may falter without a fuller and more quantitative elucidation of the physical and chemical characteristics of DNA and chromatin in vivo.

The distribution of inverted repeat sequences in the Saccharomyces cerevisiae genome.

Although a variety of possible functions have been proposed for inverted repeat sequences (IRs), it is not known which of them might occur in vivo. We investigate this question by assessing the distributions and properties of IRs in the Saccharomyces cerevisiae (SC) genome. Using the IRFinder algorithm we detect 100,514 IRs having copy length greater than 6 bp and spacer length less than 77 bp. To assess statistical significance we also determine the IR distributions in two types of randomization of the S. cerevisiae genome. We find that the S. cerevisiae genome is significantly enriched in IRs relative to random. The S. cerevisiae IRs are significantly longer and contain fewer imperfections than those from the randomized genomes, suggesting that processes to lengthen and/or correct errors in IRs may be operative in vivo. The S. cerevisiae IRs are highly clustered in intergenic regions, while their occurrence in coding sequences is consistent with random. Clustering is stronger in the 3' flanks of genes than in their 5' flanks. However, the S. cerevisiae genome is not enriched in those IRs that would extrude cruciforms, suggesting that this is not a common event. Various explanations for these results are considered.

FUBP1 and FUBP2 enforce distinct epigenetic setpoints for MYC expression in primary single murine cells.

Physiologically, MYC levels must be precisely set to faithfully amplify the transcriptome, but in cancer MYC is quantitatively misregulated. Here, we study the variation of MYC amongst single primary cells (B-cells and murine embryonic fibroblasts, MEFs) for the repercussions of variable cellular MYC-levels and setpoints. Because FUBPs have been proposed to be molecular "cruise controls" that constrain MYC expression, their role in determining basal or activated MYC-levels was also examined. Growing cells remember low and high-MYC setpoints through multiple cell divisions and are limited by the same expression ceiling even after modest MYC-activation. High MYC MEFs are enriched for mRNAs regulating inflammation and immunity. After strong stimulation, many cells break through the ceiling and intensify MYC expression. Lacking FUBPs, unstimulated MEFs express levels otherwise attained only with stimulation and sponsor MYC chromatin changes, revealed by chromatin marks. Thus, the FUBPs enforce epigenetic setpoints that restrict MYC expression.

Superhelical destabilization in regulatory regions of stress response genes.

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.

Persistence lengths of DNA obtained from Brownian dynamics simulations.

The persistence length of DNA has been studied for decades; however, experimentally obtained values of this quantity have not been entirely consistent. We report results from Brownian dynamics simulations that address this issue, validating and demonstrating the utility of an explicitly double-stranded model for mesoscale DNA dynamics. We find that persistence lengths calculated from rotational relaxation increase with decreasing ionic strength, corroborating experimental evidence, but contradicting results obtained from wormlike coil assumptions. Further, we find that natural curvature does not significantly affect the persistence length, corroborating cyclization efficiency measurements, but contradicting results from cryo-EM.

OriDB: a DNA replication origin database.

Replication of eukaryotic chromosomes initiates at multiple sites called replication origins. Replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where several complementary studies have mapped their locations genome-wide. We have collated these datasets, taking account of the resolution of each study, to generate a single list of distinct origin sites. OriDB provides a web-based catalogue of these confirmed and predicted S.cerevisiae DNA replication origin sites. Each proposed or confirmed origin site appears as a record in OriDB, with each record comprising seven pages. These pages provide, in text and graphical formats, the following information: genomic location and chromosome context of the origin site; time of origin replication; DNA sequence of proposed or experimentally confirmed origin elements; free energy required to open the DNA duplex (stress-induced DNA duplex destabilization or SIDD); and phylogenetic conservation of sequence elements. In addition, OriDB encourages community submission of additional information for each origin site through a User Notes facility. Origin sites are linked to several external resources, including the Saccharomyces Genome Database (SGD) and relevant publications at PubMed. Finally, a Chromosome Viewer utility allows users to interactively generate graphical representations of DNA replication data genome-wide. OriDB is available at www.oridb.org.

Visualization of the detailed structure of plasmid DNA.

We report results from scanning tunneling microscopy experiments in which supercoiling of the plasmid, pTNT, leads to localized duplex denaturation at a single location. Measurements of the imaged strand-separated region suggest an extent of approximately 60 base pairs (bp's), in excellent agreement with the prediction of a statistical mechanical analysis of the pTNT base sequence that accounts for the topology-mediated global coupling of melting behaviors. Notwithstanding that the sequence of pTNT includes a 30-bp run of poly-A, where thermodynamic considerations alone predict significant instability, based on the statistical result, we propose the location of denaturation is an approximately 60-bp run, spanning positions 2050-2110, that is 82% AT-rich, terminally flanks the beta-lactamase coding region, and is known to contain two sites susceptible to cleavage by the DraI restriction enzyme.