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Mouse models have revealed an intrinsic nonrandom hypermutation profile within antibody-coding exons. The somatic mutator deaminase protein, activation-induced cytidine deaminase (AID), favors DNA motifs that lie in a flexible configuration; both micro- and mesoscale preferences can shape in vivo somatic hypermutation profiles. DNA mechanics play an essential role in generating beneficial mutations in antigen-contacting residues of coding sequences. This cis -mechanical feature is conserved in antibody genes from species that use somatic hypermutation as a major diversification strategy. DNA mechanics can affect mutagenesis in AID off targets, contributing to lymphomagenesis. During antibody affinity maturation, beneficial mutations preferentially occur in DNA regions encoding antigen-contacting amino acids. The nonrandomness of predisposed mutations has long been a mystery in immunology. Mouse models indicate a mysterious cis element in DNA sequences that contributes to sequence-dependent DNA flexibility, with the latter being selected during the evolution of adaptive immunity. This is relevant because coding sequences can play a noncoding role through DNA mechanics, a code which might harbor hidden genetic information. Antibody-coding genes accumulate somatic mutations to achieve antibody affinity maturation. Genetic dissection using various mouse models has shown that intrinsic hypermutations occur preferentially and are predisposed in the DNA region encoding antigen-contacting residues. The molecular basis of nonrandom/preferential mutations is a long-sought question in the field. Here, we summarize recent findings on how single-strand (ss)DNA flexibility facilitates activation-induced cytidine deaminase (AID) activity and fine-tunes the mutation rates at a mesoscale within the antibody variable domain exon. We propose that antibody coding sequences are selected based on mutability during the evolution of adaptive immunity and that DNA mechanics play a noncoding role in the genome. The mechanics code may also determine other cellular DNA metabolism processes, which awaits future investigation. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis, the causative agent of tuberculosis, has evolved over time into a multidrug resistance strain that poses a serious global pandemic health threat. The ability to survive and remain dormant within the host macrophage relies on multiple transcription factors contributing to virulence. To date, very limited structural insights from crystallographic and NMR studies are available for TFs and TF–DNA binding events. Understanding the role of DNA structure in TF binding is critical to deciphering MTB pathogenicity and has yet to be resolved at the genome scale. In this work, we analyzed the compositional and conformational preference of 21 mycobacterial TFs, evident at their DNA binding sites, in local and global scales. Results suggest that most TFs prefer binding to genomic regions characterized by unique DNA structural signatures, namely, high electrostatic potential, narrow minor grooves, high propeller twist, helical twist, intrinsic curvature, and DNA rigidity compared to the flanking sequences. Additionally, preference for specific trinucleotide motifs, with clear periodic signals of tetranucleotide motifs, are observed in the vicinity of the TF– DNA interactions. Altogether, our study reports nuanced DNA shape and structural preferences of 21 TFs. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis, the causative agent of tuberculosis, has evolved over time into a multidrug resistance strain that poses a serious global pandemic health threat. The ability to survive and remain dormant within the host macrophage relies on multiple transcription factors contributing to virulence. To date, very limited structural insights from crystallographic and NMR studies are available for TFs and TF–DNA binding events. Understanding the role of DNA structure in TF binding is critical to deciphering MTB pathogenicity and has yet to be resolved at the genome scale. In this work, we analyzed the compositional and conformational preference of 21 mycobacterial TFs, evident at their DNA binding sites, in local and global scales. Results suggest that most TFs prefer binding to genomic regions characterized by unique DNA structural signatures, namely, high electrostatic potential, narrow minor grooves, high propeller twist, helical twist, intrinsic curvature, and DNA rigidity compared to the flanking sequences. Additionally, preference for specific trinucleotide motifs, with clear periodic signals of tetranucleotide motifs, are observed in the vicinity of the TF–DNA interactions. Altogether, our study reports nuanced DNA shape and structural preferences of 21 TFs.

Atomic force microscopy (AFM) was used to conduct single‐molecule imaging of protein/DNA complexes involved in the regulation of the arabinose operon of Escherichia coli. In the presence of arabinose, the transcription regulatory protein AraC binds to a 38 bp region consisting of the araI1 and araI2 half‐sites. The domain positioning of full‐length AraC, when bound to DNA, was not previously known. In this study, AraC was combined with 302 and 560 bp DNA and arabinose, deposited on a mica substrate, and imaged with AFM in air. High resolution images of 560 bp DNA, where bound protein was visible, showed that AraC induces a bend in the DNA with an angle 60° ± 12° with a median of 55°. These results are consistent with earlier gel electrophoresis measurements that measured the DNA bend angle based on migration rates. By using known domain structures of AraC, geometric constraints, and contacts determined from biochemical experiments, we developed a model of the tertiary and quaternary structure of DNA‐bound AraC in the presence of arabinose. The DNA bend angle predicted by the model is in agreement with the measurement values. We discuss the results in view of other regulatory proteins that cause DNA bending and formation of the open complex to initiate transcription. [ABSTRACT FROM AUTHOR]

Cell fate decisions are controlled by sequence-specific transcription factors (TFs), referred to as 'pioneer' factors, that bind their target sites within nucleosomes ('pioneer binding') and thus initiate chromatin opening. However, pioneers bind just a minority of their recognition sequences present in the genome, suggesting that local sequence context features may regulate pioneer binding. Here, we developed PIONEAR-seq, a highly parallel sequencing-based biochemical assay for high-throughput analysis of TF binding to nucleosomes on nucleosome positioning sequences. Using PIONEAR-seq, we characterized the pioneer binding of 7 human pioneer TFs. Comparison of TF binding to nucleosomes based on the synthetic Widom 601 (W601) model sequence versus three different genomic sequences revealed that the positional preferences of these TFs' binding to nucleosomes ( i.e. , dyad, periodic and end binding) is determined by the broader sequence context of the nucleosome, rather than being a property intrinsic to the TF. We propose a model where the flexibility and rigidity within nucleosomal DNA regulate where pioneers bind within nucleosomes. Our results suggest that the broader physical properties of nucleosomal DNA represent another layer of cis -regulatory information read out by TFs in eukaryotic genomes., Competing Interests: Competing Interests All authors declare no competing interests.

Nucleoid‐associated proteins (NAPs) maintain bacterial nucleoid configuration through their architectural properties of DNA bending, wrapping, and bridging. However, the contribution of DNA structural alterations to DNA‐NAP recognition at the genomic scale remains unresolved. Present work dissects the DNA sequence, shape and altered structural preferences at a genomic scale for six NAPs in Mycobacterium tuberculosis. Results suggest narrower minor groove width (MGW) and higher DNA rigidity are marked for the binding sites of EspR and Lsr2, while mIHF, MtHU and NapM have heterogeneous DNA structural predilections. In contrast, WhiB4–DNA‐binding sites were characterized by wider MGW, highly deformable and less curved DNA. This work provides systematic insight into NAP‐mediated genome organization as a function of DNA structural features. [ABSTRACT FROM AUTHOR]

Abstract Ru(bpy)2dppz2+ has been widely used as a probe for exploring the structure of double-stranded DNA (dsDNA). The flexibility change of DNA helix is important in many of its biological functions but not well understood. Here, flexibility change of dsDNA helix caused by intercalation with Ru(bpy)2dppz2+ was investigated using the atomic force microscopy. At first, the interactions between ruthenium complex and dsDNA helix were characterized and the binding site size (p = 2.87 bp) and binding constant (Ka = 5.9 * 107 M−1) were determined by the relative extension of DNA helix using the equation of McGhee and von Hippel. By measuring intercalator-induced DNA elongation and the mean square of end-to-end distance at different molar ratios of Ru(bpy)2dppz2+ to dsDNA, the changes of persistence length under different ruthenium concentrations were determined by the worm-like chain model. We found that the persistence length of dsDNA decreased with increasing Ru(bpy)2dppz2+ concentration, demonstrating that the flexibility of dsDNA obviously enhanced due to the intercalation. Especially, the persistence length changed greatly from 54 to 34 nm on changing the molar ratio of ruthenium to dsDNA from 0 to 0.2. We speculated that the intercalation of dsDNA with Ru(bpy)2dppz2+ resulted in local deformation or bending of the DNA duplex. In addition, the thermal dynamic stability of DNA helix was measured with high resolution melting method which revealed the increase in thermal dynamic stability of DNA helix due to the ruthenium intercalation.

Abstract: Bent DNA, or DNA that is locally more flexible, is a recognition motif for many DNA binding proteins. These DNA conformational properties can thus influence many cellular processes, such as replication, transcription, and DNA repair. The importance of these DNA conformational properties is juxtaposed to the experimental difficulty to accurately determine small bends, locally more flexible DNA, or a combination of both (bends with increased flexibility). In essence, many current bulk methods use average quantities, such as the average end‐to‐end distance, to extract DNA conformational properties; they cannot access the additional information that is contained in the end‐to‐end distance distributions. We developed a method that exploits this additional information to determine DNA conformational parameters. The method is based on matching end‐to‐end distance distributions obtained experimentally by atomic force microscopy imaging to distributions obtained from simulations. We applied this method to investigate cisplatin GG biadducts. We found that cisplatin induces a bend angle of 36° and softens the DNA locally around the bend. [ABSTRACT FROM AUTHOR]

Other than being a carrier of the code of life deoxyribonucleic acid (DNA) can also be used as a kind of ideal biomaterial with good biocompatibility. The basis of DNA dynamic nanotechnology is the toehold-mediated strand displacement reactions. Utilizing the specificity and predictability of Watson–Crick base pairing, and the programmability of the base sequence of DNA, researchers can construct the molecular machine and precisely control its operation to realize various complex molecular calculations. In this paper, we reviewed the progress of DNA structure and mechanical properties in recent years, discussed the microcosmic mechanism of DNA strand replacement reaction, and introduced the latest achievements in DNA molecular computing and its application in DNA constant temperature self-assembly. [ABSTRACT FROM AUTHOR]

A chromatosome consisting of a nucleosome core, linker DNA, and linker histone (LH) is an important structural element of chromatin and plays a role in replication and transcription regulation. There are two experimentally confirmed modes of LH binding to the nucleosome and linker DNA that differ in their geometry: on-dyad and off-dyad binding. It was shown that the LH amino acid sequence influences the type of histone binding and the conformation of the chromatosome. However, the geometry of linker DNA bound with LH also changes. Thus, the mutual influence of these factors and the molecular basis determining the type of LH binding to nucleosomes remain unclear. In this study, molecular modeling methods, including homology modeling, atom-atom interaction analysis, and DNA deformation energy analysis, were applied to study the joint effect of the LH amino acid sequence and the DNA nucleotide sequence on the configuration of the chromatosome. The known crystal and NMR structures of the chromatosome for atom-atom interactions of LH and DNA, as well as the energy of DNA deformation in these structures for various DNA sequences, were analyzed. For various LH H1 variants, the analysis was carried out using homology modeling methods. Sequence-dependent differences in the bending energy of the linker DNA for two different conformations of the chromatosome were found, and nucleotide sequences preferred for these structures were proposed. As a result of the analysis, it was shown that the DNA nucleotide sequence, along with the LH amino acid sequence, influences the type of binding to the nucleosome. Hypotheses for experimental verification have been formulated, according to which the type of LH binding can change with different DNA nucleotide sequences. [ABSTRACT FROM AUTHOR]

The twisting deformation of mechanically stretched DNA molecules is studied by a coarse grained Hamiltonian model incorporating the fundamental interactions that stabilize the double helix and accounting for the radial and angular base pair fluctuations. The latter are all the more important at short length scales in which DNA fragments maintain an intrinsic flexibility. The presented computational method simulates a broad ensemble of possible molecule conformations characterized by a specific average twist and determines the energetically most convenient helical twist by free energy minimization. As this is done for any external load, the method yields the characteristic twist-stretch profile of the molecule and also computes the changes in the macroscopic helix parameters i.e. average diameter and rise distance. It is predicted that short molecules under stretching should first over-twist and then untwist by increasing the external load. Moreover, applying a constant load and simulating a torsional strain which over-twists the helix, it is found that the average helix diameter shrinks while the molecule elongates, in agreement with the experimental trend observed in kilo-base long sequences. The quantitative relation between percent relative elongation and superhelical density at fixed load is derived. The proposed theoretical model and computational method offer a general approach to characterize specific DNA fragments and predict their macroscopic elastic response as a function of the effective potential parameters of the mesoscopic Hamiltonian. [ABSTRACT FROM AUTHOR]

Recently we have developed a new method for studying DNA based on ultrasound - induced cleavage of DNA sugar-phosphate backbone. Relative cleavage rates of the phosphodiester bonds in all 16 dinucleotides have been determined. The increased amount of data sampling (of more than 20 000 nucleotides) made it also possible to obtain cleavage rates in all 256 possible tetranucleotides. These values quantitatively characterize sequence effects on conformational dynamics of DNA sugar phosphate backbone. Same type of DNA heterogeneity have been discovered and studied using its chemical cleavage induced by various chemical agents and DNAse I. The presence of essential heterogeneity in structural properties of DNA might be a key for physical mapping of the genomes, i.e. determining the structural profiles being responsible for DNA recognition by gene expression regulation machinery.

We use Förster Resonant Energy Transfer (FRET) in order to measure the increase of flexibility of short ds-DNA induced by the intercalation of dipyridophenazine (dppz) ligand in between DNA base pairs. By using a DNA double strand fluorescently labeled at its extremeties, it is shown that the end-to-end length increase of DNA due to the intercalation of one dppz ligand is smaller than the DNA base pair interdistance. This may be explained either by a local bending of the DNA or by an increase of its flexibility. The persistence length of the formed DNA/ligand is evaluated. The described structure may have implications in the photophysical damages induced by the complexation of DNA by organometallic molecules.

Background: Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joining and generates a short (4-6 bp) duplication of host DNA flanking the provirus. We previously determined two key parameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiency virus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integration sites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase. Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings with respect to length of target site duplication (TSD). Results: Integration datasets covering six viral genera and the three lengths of TSD were accessed from the literature or generated in this work. All viruses exhibited significant enrichments of flexible YR and/or selection against rigid RY dinucleotide steps at the centers of integration sites, and the magnitude of this enrichment inversely correlated with TSD length. The DNA sequence environments of in vivo-generated HIV-1 and PFV sites were consistent with integration into nucleosomes, however, the local sequence preferences were largely independent of target DNA chromatinization. Integration sites derived from cells infected with the gammaretrovirus reticuloendotheliosis virus strain A (Rev-A), which yields a 5 bp TSD, revealed the targeting of global chromatin features most similar to those of Moloney murine leukemia virus, which yields a 4 bp duplication. In vitro assays revealed that Rev-A integrase interacts with and is catalytically stimulated by cellular bromodomain containing 4 protein. Conclusions: Retroviral integrases have likely evolved to bend target DNA to fit scissile phosphodiester bonds into two active sites for integration, and viruses that cut target DNA with a 6 bp stagger may not need to bend DNA as sharply as viruses that cleave with 4 bp or 5 bp staggers. For PFV and HIV-1, the selection of signature bases and central flexibility at sites of integration is largely independent of chromatin structure. Furthermore, global Rev-A integration is likely directed to chromatin features by bromodomain and extraterminal domain proteins. [ABSTRACT FROM AUTHOR]

The Cre/loxP system is widely used as a genetic tool to manipulate DNA. Cre recombinase catalyzes site-specific recombination between 34 bp loxP sites. Each loxP site is recognized by two Cre molecules assuming a cleaving (Cre) and non-cleaving (Cre) activity. Despite the symmetry in the sequences of the arms of loxP, available biochemical data show strong evidence that the recombination reaction is asymmetric with a preferred strand exchange order. The asymmetry comes from the spacer separating the two sets of palindromic arms of the loxP sequence. However, it remains to be understood how this preferential order is established. We apply computational structure-based methods and perform a thorough detailed analysis of available structural and biochemical information on the Cre/loxP system in order to investigate such asymmetry in the recombination, and we propose a rationale to explain the determinants favoring the strand exchange order. We show that the structural properties of the DNA flanking sequence of the spacer guide the recombination, and we establish the role of residues R118, R121 and K122 from Cre, which contact the spacer region and by clamping the DNA inhibit the cleavage on the second arm of loxP. Our studies give an atomistic insight on the synapsis state of the recombination process in the Cre/loxP system and highlight the importance of the flexibility and other intrinsic properties of the flanking regions of the DNA spacer to establish a preferential strand exchange order. [ABSTRACT FROM AUTHOR]

In 25-30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification. [ABSTRACT FROM AUTHOR]

Nucleosome positioning plays an essential role in various fundamental cellular processes by modulating the accessibility of DNA to binding proteins. Understanding the mechanisms and precise recognition of nucleosome positioning along genomic sequences are substantially important for elucidating regulations of cellular processes such as DNA replication, recombination, and gene transcription. In this report, we present a knowledge-based model for calculation of deformational energy of nucleosomal DNA and apply the model to the prediction of nucleosome positioning along the genome of Saccharomyces cerevisiae accurately. The model successfully predicted genome-wide in vitro nucleosome positions. When combined with quadratic discriminant classifier, the model achieved an accuracy of 92.9 % in discriminating in vitro nucleosome forming sequences from nucleosome inhibiting sequences. Our result supports the debated notion that the nucleosome positioning in the genomic sequences is guided primarily by deformational properties of DNA. [ABSTRACT FROM AUTHOR]

Despite our long familiarity with how the genetic code specifies the amino acid sequence, we still know little about why it is organized in the way that it is. Contrary to the view that the organization of the genetic code is a “frozen accident” of evolution, recent studies have demonstrated that it is highly nonrandom, with implications for both codon assignment and usage. We hypothesize that this inherent nonrandomness may facilitate the coexistence of both sequence and structural information in DNA. Here, we take advantage of a simple metric of intrinsic DNA flexibility to analyze mutational effects on the four phosphate linkages present in any given codon. Application of a simple evolutionary neutral model of substitution to random sequences, translated with alternative genetic codes, reveals that the standard code is highly optimized to favor synonymous substitutions that maximize DNA polymer flexibility, potentially counteracting neutral evolutionary drift toward stiffer DNA caused by spontaneous deamination. Comparison to existing mutational patterns in yeast also demonstrates evidence of strong selective constraint on DNA flexibility, especially at so-called “silent” sites. We also report a fundamental relationship between DNA flexibility, codon usage bias, and several important evolutionary descriptors of comparative genomics (e.g., base composition, transition/transversion ratio, and nonsynonymous vs. synonymous substitution rate). Recent advances in structural genomics have emphasized the role of the DNA polymer's flexibility in both gene function and whole genome folding, thereby implicating possible reasons for codons to facilitate the multiplexing of both genetic and structural information within the same molecular context. [ABSTRACT FROM AUTHOR]

Abstract: Understanding and predicting the mechanical properties of protein/DNA complexes are challenging problems in biophysics. Certain architectural proteins bind DNA without sequence specificity and strongly distort the double helix. These proteins rapidly bind and unbind, seemingly enhancing the flexibility of DNA as measured by cyclization kinetics. The ability of architectural proteins to overcome DNA stiffness has important biological consequences, but the detailed mechanism of apparent DNA flexibility enhancement by these proteins has not been clear. Here, we apply a novel Monte Carlo approach that incorporates the precise effects of protein on DNA structure to interpret new experimental data for the bacterial histone-like HU protein and two eukaryotic high-mobility group class B (HMGB) proteins binding to ∼200-bp DNA molecules. These data (experimental measurement of protein-induced increase in DNA cyclization) are compared with simulated cyclization propensities to deduce the global structure and binding characteristics of the closed protein/DNA assemblies. The simulations account for all observed (chain length and concentration dependent) effects of protein on DNA behavior, including how the experimental cyclization maxima, observed at DNA lengths that are not an integral helical repeat, reflect the deformation of DNA by the architectural proteins and how random DNA binding by different proteins enhances DNA cyclization to different levels. This combination of experiment and simulation provides a powerful new approach to resolve a long-standing problem in the biophysics of protein/DNA interactions. [Copyright &y& Elsevier]

Abstract: This paper presents a top-down strategy to detect features in genomic sequences. The strategy''s core is to exploit dictionary-based compression algorithms and analyse the content of the automatically generated dictionary. We classify the different over-represented segments and in the case study we correlate them to experimentally identified or theoretically forecasted biological features. A large spectrum analysis reveals that the only feature co-located with the a priori extracted segments is the torsional flexibility of DNA, while non-B DNA configurations are anti-localized and other features are mostly independent of the extracted sequences. This analysis unravels complex relationships between the linguistic structures investigated under our approach and some known biological features. [Copyright &y& Elsevier]

Abstract: Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3′ phosphate linkage flexibility, monitored by 31P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA–protein complexes. In sum, this work demonstrates that specificity in DNA–DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes. [Copyright &y& Elsevier]

Persistence length is the foremost measure of DNA flexibility. Its origins lie in polymer theory which was adapted for DNA following the determination of B-DNA structure in 1953. There is no single definition of persistence length used, and the links between published definitions are based on assumptions which may, or may not be, clearly stated. DNA flexibility is affected by local ionic strength, solvent environment, bound ligands and intrinsic sequence-dependent flexibility. This article is a review of persistence length providing a mathematical treatment of the relationships between four definitions of persistence length, including: correlation, Kuhn length, bending, and curvature. Persistence length has been measured using various microscopy, force extension and solution methods such as linear dichroism and transient electric birefringence. For each experimental method a model of DNA is required to interpret the data. The importance of understanding the underlying models, along with the assumptions required by each definition to determine a value of persistence length, is highlighted for linear dichroism data, where it transpires that no model is currently available for long DNA or medium to high shear rate experiments. [ABSTRACT FROM AUTHOR]

Stacking energy of all the 10 unique DNA base-pair steps (bp step) are calculated using density functional theory within the ultrasoft pseudopotential plane wave method and local density approximation for the exchange-correlation functional. We have studied the dependence of stacking energy on twist angle, an aspect found difficult to explain using classical theory. We have found that the twist angle for different bp steps at stacking energy minimum matches extremely well with the values of average twist obtained from B-DNA crystal structure data. This indicates that the use of a proper quantum chemical method to calculate the π-π electronic interactions may explain stacking energy without incorporating hydrophobic interaction through solvent or effect of backbone through pseudobond. From the twist angle-dependent stacking energy profile, we have also generated the probability distributions of twist for all the bp steps and calculated the variance of the distribution. Our calculated variances show similar trend to that of the experimental data for which sufficient numbers of data are available. The TA, AT, and CG doublets show large variances among the 10 possible bp steps, indicating their maximum flexibility. This might be the case of unusual deformation observed at the TATA-box while binding to TBP protein. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008 [ABSTRACT FROM AUTHOR]

Abstract: DNA is not only a nucleotide sequence which allows the binding of regulators but its intrinsic structural properties such as curvature and flexibility are also viewed as playing an active role in the regulation of transcription. Our combination of computer modelling and AFM imaging allow direct access to DNA curvature and flexibility. We have searched for these DNA structural features involved in transcription regulation within the IL-2Rα gene promoter. Investigation of these structural characteristics shows concordant results. First, in the core promoter, the region containing the functional TATA box shows intrinsic curvature associated with a peculiar distribution of flexibility. Both these inherent properties are characteristic of this region as compared with the other parts of the promoter. Second, the proximal promoter exhibits two important regions: a first one flexible and curved, followed by a segment of rigid linear DNA, each localised within one of the two Positive Regulatory Regions PRRI and PRRII respectively. Based on these observations, we propose different roles for DNA curvature and/or flexibility in promoter sequences. [Copyright &y& Elsevier]

Abstract: Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays. [Copyright &y& Elsevier]

Abstract: The search for the genetic variations underlying all human phenotypes is in its infancy but must be one of the long term goals of the scientific community. There is evidence that most, if not all human phenotypes, including illnesses are influenced by the genetic makeup of the individual. There are an estimated 11 million human genetic polymorphisms with a minor allele frequency >1% and possibly many times that number of rare sequence variants. The proportion of these sequence variants which have any functional effect is unknown but it is likely that the majority of those which influence illness lie outside of the amino acid coding regions of genes, and affect the regulation of gene expression—these are called rSNPs. Recent research suggests that about 50% of genes have one or more common rSNPs associated with them and probably most if not all genes have an rSNP within the human population. In the long term, determining which polymorphisms are potentially functional must be done bio-informatically using algorithms based upon experimental data. However, at the current time, the limited data that has been obtained does not allow the creation of such an algorithm. In vitro studies suggest that a large proportion of rSNPs lie within the core and proximal promoter regions of genes but it is not clear how the majority of these influence transcription, as they do not appear to be within any known transcription factor binding sites. However, promoter regions possess a number of sequence-dependent characteristics which make them distinct from the rest of the genome, namely stability, curvature and flexibility. Subtle changes to these features may underlie the mechanisms by which many polymorphisms exert their function. [Copyright &y& Elsevier]

Abstract: Experimental evidence suggests DNA mechanical properties, in particular intrinsic curvature and flexibility, have a role in many relevant biological processes. Systematic investigations about the origin of DNA curvature and flexibility have been carried out; however, most of the applied experimental techniques need simplifying models to interpret the data, which can affect the results. Progress in the direct visualization of macromolecules allows the analysis of morphological properties and structural changes of DNAs directly from the digitised micrographs of single molecules. In addition, the statistical analysis of a large number of molecules gives information both on the local intrinsic curvature and the flexibility of DNA tracts at nanometric scale in relatively long sequences. However, it is necessary to extend the classical worm-like chain model (WLC) for describing conformations of intrinsically straight homogeneous polymers to DNA. This review describes the various methodologies proposed by different authors. [Copyright &y& Elsevier]

Using fluorescence polarization anisotropy (FPA), we measured the torsional constant of various DNA oligomers in different sequences and calculated the value for each of the 136 unique tetranucleotides. From these values, we obtained a «rigidity profile» for every double-stranded DNA sequence. We tested the code in the analysis of DNA sequences able to form nucleosomes. More than 50% of the sequences studied showed a common 20 and/or 30 bp modulation of the torsional constant. Many other profiles of rigidity were observed in the remaining sequences and this variety in torsional constant modulation may be related to functional differences between nucleosomes. [Copyright &y& Elsevier]

A database of the structural properties of all 32,896 unique DNA octamer sequences has been calculated, including information on stability, the minimum energy conformation and flexibility. The contents of the database have been analysed using a variety of Euclidean distance similarity measures. A global comparison of sequence similarity with structural similarity shows that the structural properties of DNA are much less diverse than the sequences, and that DNA sequence space is larger and more diverse than DNA structure space. Thus, there are many very different sequences that have very similar structural properties, and this may be useful for identifying DNA motifs that have similar functional properties that are not apparent from the sequences. On the other hand, there are also small numbers of almost identical sequences that have very different structural properties, and these could give rise to false-positives in methods used to identify function based on sequence alignment. A simple validation test demonstrates that structural similarity can differentiate between promoter and non-promoter DNA. Combining structural and sequence similarity improves promoter recall beyond that possible using either similarity measure alone, demonstrating that there is indeed information available in the structure of double-helical DNA that is not readily apparent from the sequence. [Copyright &y& Elsevier]

The 5′ d-TpG 3′ element is a part of DNA sequences involved in regulation of gene expression and is also a site for intercalation of several anticancer drugs. Solution conformation of DNA duplex d-TGATCA containing this element has been investigated by two-dimensional NMR spectroscopy. Using a total of 12 torsional angles and 121 distance constraints, structural refinement has been carried out by restrained molecular dynamics (rMDs) in vacuum up to 100 ps. The structure is characterized by a large positive roll at TpG/CpA base pair step and large negative propeller twist for AT and TA base pairs. The backbone torsional angle, γ(O5′–C5′–C4′–C3′), of T1 residue adopts a trans-conformation which is corroborated by short intra nucleotide T1H6–T1H5′ (3.7 Å) distance in nuclear overhauser effect spectroscopy (NOESY) spectra while the backbone torsional angle, β(P–O5′–C5′–C4′), exists in trans as well as gauche state for T1 and C5 residues. There is evidence of significant flexibility of the sugar–phosphate backbone with rapid inter-conversion between two different conformers at TpG/CpA base pair step. The base sequence dependent variations and local structural heterogeneity have important implications in specific recognition of DNA by ligands. [Copyright &y& Elsevier]

The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein''s DNA-binding site modulate the affinity of DNA for protein. The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components. We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor–OR1 complex requires the presence of monovalent cations; however, repressor–OR3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from ⋅OH radical cleavage. Altering the relative length of the poly(dA)·poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressor''s affinities for these two sites. Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins. [Copyright &y& Elsevier]

The histone-like protein HU from Escherichia coli is involved in DNA compaction and in processes such as DNA repair and recombination. Its participation in these events is reflected in its ability to bend DNA and in its preferred binding to DNA junctions and DNA with single-strand breaks. Deinococcus radiodurans is unique in its ability to reconstitute its genome from double strand breaks incurred after exposure to ionizing radiation. Using electrophoretic mobility shift assays (EMSA), we show that D. radiodurans HU (DrHU) binds preferentially only to DNA junctions, with half-maximal saturation of 18 nM. In distinct contrast to E. coli HU, DrHU does not exhibit a marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able to mediate circularization of linear duplex DNA. These unexpected properties identify DrHU as the first member of the HU protein family not to serve an architectural role and point to its potential participation in DNA recombination events. Our data also point to a mechanism whereby differential target site selection by HU proteins is achieved and suggest that the substrate specificity of HU proteins should be expected to vary as a consequence of their individual capacity for inducing the required DNA bend. [Copyright &y& Elsevier]

In spite of the knowledge of the nucleosome molecular structure, the role of DNA intrinsic curvature in determining nucleosome stabilization is still an open question. In this paper, we describe a general model that allows the prediction of the nucleosome stability, tested on 83 different DNA sequences, in surprising good agreement with the experimental data, carried out in ours as well as in many other laboratories. The model is based on the dual role of DNA curvature in nucleosome thermodynamic stabilization. A critical test is the evaluation of the nucleosome free energy relative to a Crithidia fasciculata kinetoplast DNA fragment, which represents the most curved DNA found so far in biological systems and, therefore, is generally believed to form a highly stable nucleosome. [Copyright &y& Elsevier]

We have constructed the potential energy surfaces for all unique tetramers, hexamers and octamers in double helical DNA, as a function of the two principal degrees of freedom, slide and shift at the central step. From these potential energy maps, we have calculated a database of structural and flexibility properties for each of these sequences. These properties include: the values of each of the six step parameters (twist roll, tilt, rise, slide and shift), for each step of the sequence; flexibility measures for both decrease and increase in each property value from the minimum energy conformation for the central step; and the deviation from the path of a hypothetical straight octamer. In an analysis of structural change as a function of sequence length, we observe that almost all DNA tends to B-DNA and becomes less flexible. A more detailed analysis of octamer properties has allowed us to determine the structural preferences of particular sequence elements. GGC and GCC sequences tend to confer bistability, low stability and a predisposition to A-form DNA, whereas AA steps strongly prefer B-DNA and inhibit A-structures. There is no correlation between flexibility and intrinsic curvature, but bent DNA is less stable than straight. The most difficult deformation is undertwisting. The TA step stands out as the most flexible sequence element with respect to decreasing twist and increasing roll. However, as with the structural properties, this behavior is highly context-dependent and some TA steps are very straight. [Copyright &y& Elsevier]

DNA is an extensible molecule, and an extended conformation of DNA is involved in some biological processes. We have examined the effect of elongation stress on the conformational properties of DNA base pairs by conformational analysis. The calculations show that stretching does significantly affect the conformational properties and flexibilities of base pairs. In particular, we have found that the propeller twist in base pairs reverses its sign upon stretching. The energy profile analysis indicates that electrostatic interactions make a major contribution to the stabilization of the positive-propeller-twist configuration in stretched DNA. This stretching also results in a monotonic decrease in the helical twist angle, tending to unwind the double helix. Fluctuations in most variables initially increase upon stretching, because of unstacking of base pairs, but then the fluctuations decrease as DNA is stretched further, owing to the formation of specific interactions between base pairs induced by the positive propeller twist. Thus, the stretching of DNA has particularly significant effects upon DNA flexibility. These changes in both the conformation and flexibility of base pairs probably have a role in functional interactions with proteins. [ABSTRACT FROM AUTHOR]

p53 is a 53 kDa nuclear phosphoprotein. Its function as a tumor suppressor critically lies in its ability to recognize its target DNA response elements as a tetramer. Here, we report the structural theme intrinsic to the response element DNA that governs this recognition phenomenon. The intrinsic flexibility or dynamic bending between two distinctly different, but naturally occurring p53 response elements has been compared by ring closure. Results show that DNA binding sites containing helically phased d(CATG.CATG) tetra-nucleotide sequences at the centers of quasi-dyad symmetry in each half-response site are more intrinsically flexible (i.e. preferentially bent under axial stress) than their d(CTTG.CTTG) counterparts. Intriguingly, p53 binding sites containing these more flexible d(CATG.CATG) sequence elements also exhibit a stronger tendency for tetrameric binding of the p53 DNA binding domain peptide. Examination of the shapes of DNA microcircles obtained by circularization of oligomers constructed from such flexible p53 target DNA sequences in tandem using MacMode atomic force microscopy directly revealed sequence-specific kinks in solution. The tetra-nucleotide sequence d(CATG.CATG) is highly conserved in most functional p53 response elements. Consequently, we propose that the sequence-specific kinks originating from d(CATG.CATG) sequences could be a common structural theme in p53 response elements and as evident from the results reported here, could be a determinant of binding site recognition by the p53 protein and the subsequent stability of the p53–DNA complex. [Copyright &y& Elsevier]

The persistence length of DNA, a, depends both on the intrinsic curvature of the double helix and on the thermal fluctuations of the angles between adjacent base-pairs. We have evaluated two contributions to the value of a by comparing measured values of a for DNA containing a generic sequence and for an “intrinsically straight” DNA. In each 10 bp segment of the intrinsically straight DNA an initial sequence of five bases is repeated in the sequence of the second five bases, so any bends in the first half of the segment are compensated by bends in the opposite direction in the second half. The value of a for the latter DNA depends, to a good approximation, on thermal fluctuations only; there is no intrinsic curvature. The values of a were obtained from measurements of the cyclization efficiency for short DNA fragments, about 200 bp in length. This method determines the persistence length of DNA with exceptional accuracy, due to the very strong dependence of the cyclization efficiency of short fragments on the value of a. We find that the values of a for the two types of DNA fragment are very close and conclude that the contribution of the intrinsic curvature to a is at least 20 times smaller than the contribution of thermal fluctuations. The relationship between this result and the angles between adjacent base-pairs, which specify the intrinsic curvature, is analyzed. [Copyright &y& Elsevier]

A theoretical model for predicting intrinsic and induced DNA superstructures as well as their thermodynamic properties is presented. Intrinsic sequence-dependent superstructures are evaluated by integrating local deviations from the canonical B-DNA of the different dinucleotide steps. Induced superstructures are obtained by adopting the principle of minimum deformation free energy, evaluated in the Fourier space, in the framework of first-order elasticity. Finally dinucleotide stacking energies and melting temperatures are considered to account for local flexibility. In fact the two scales are strongly correlated. The model works very satisfactorily in predicting the sequence-dependent effects on the DNA experimental behavior, such as the gel electrophoresis retardation, the writhe transitions in topologically constrained domains, the thermodynamic constants of circularization reactions as well as the nucleosome thermodynamic stability constants. [Copyright &y& Elsevier]

• The high-affinity binding of p53 to its responsive elements is strongly qualified by the DNA structure context. • High-affinity p53 sites and the genomic sequence environment surrounding the REs has higher axial and torsional flexibility. • The p53 cistrome gene promoters have distinct promoter architecture compared to all human promoters. The p53 tumor suppressor protein maintains the genome fidelity and integrity by modulating several cellular activities. It regulates these events by interacting with a heterogeneous set of response elements (REs) of regulatory genes in the background of chromatin configuration. At the p53-RE interface, both the base readout and torsional-flexibility of DNA account for high-affinity binding. However, DNA structure is an entanglement of a multitude of physicochemical features, both local and global structure should be considered for dealing with DNA-protein interactions. The goal of current research work is to conceptualize and abstract basic principles of p53-RE binding affinity as a function of structural alterations in DNA such as bending, twisting, and stretching flexibility and shape. For this purpose, we have exploited high throughput in-vitro relative affinity information of responsive elements and genome binding events of p53 from HT-Selex and ChIP-Seq experiments respectively. Our results confirm the role of torsional flexibility in p53 binding, and further, we reveal that DNA axial bending, stretching stiffness, propeller twist, and wedge angles are intimately linked to p53 binding affinity when compared to homeodomain, bZIP, and bHLH proteins. Besides, a similar DNA structural environment is observed in the distal sequences encompassing the actual binding sites of p53 cistrome genes. Additionally, we revealed that p53 cistrome target genes have unique promoter architecture, and the DNA flexibility of genomic sequences around REs in cancer and normal cell types display major differences. Altogether, our work provides a keynote on DNA structural features of REs that shape up the in-vitro and in-vivo high-affinity binding of the p53 transcription factor. [ABSTRACT FROM AUTHOR]

We propose an index of DNA homogeneity (IDH) based on a binary distribution model that quantifies structural and thermodynamic aggregates present in DNA primary structures. Extensive analysis of sequence databases with the IDH uncovers significant constraints on DNA sequence other than those derived from codon usage or protein function. This index clearly distinguishes between organisms of different evolutive origins and places them in disjoint domains of DNA sequence space. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman scattering (SERS)-based biosensors are promising tools for virus nucleic acid detection. However, it remains challenging for SERS-based biosensors using a sandwiching strategy to detect long-chain nucleic acids such as nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because the extension of the coupling distance (CD) between the two tethered metallic nanostructures weakens electric field and SERS signals. Herein, we report a magnetic-responsive substrate consisting of heteoronanostructures that controls the CD for ultrasensitive and highly selective detection of the N gene of SARS-CoV-2. Significantly, our findings show that this platform reversibly shortens the CD and enhances SERS signals with a 10-fold increase in the detection limit from 1 fM to 100 aM, compared to those without magnetic modulation. The optical simulation that emulates the CD shortening process confirms the CD-dependent electric field strength and further supports the experimental results. Our study provides new insights into designing a stimuli-responsive SERS-based platform with tunable hot spots for long-chain nucleic acid detection.

Ru(bpy)2dppz2+ has been widely used as a probe for exploring the structure of double-stranded DNA (dsDNA). The flexibility change of DNA helix is important in many of its biological functions but not well understood. Here, flexibility change of dsDNA helix caused by intercalation with Ru(bpy)2dppz2+ was investigated using the atomic force microscopy. At first, the interactions between ruthenium complex and dsDNA helix were characterized and the binding site size (p = 2.87 bp) and binding constant (Ka = 5.9 * 107 M−1) were determined by the relative extension of DNA helix using the equation of McGhee and von Hippel. By measuring intercalator-induced DNA elongation and the mean square of end-to-end distance at different molar ratios of Ru(bpy)2dppz2+ to dsDNA, the changes of persistence length under different ruthenium concentrations were determined by the worm-like chain model. We found that the persistence length of dsDNA decreased with increasing Ru(bpy)2dppz2+ concentration, demonstrating that the flexibility of dsDNA obviously enhanced due to the intercalation. Especially, the persistence length changed greatly from 54 to 34 nm on changing the molar ratio of ruthenium to dsDNA from 0 to 0.2. We speculated that the intercalation of dsDNA with Ru(bpy)2dppz2+ resulted in local deformation or bending of the DNA duplex. In addition, the thermal dynamic stability of DNA helix was measured with high resolution melting method which revealed the increase in thermal dynamic stability of DNA helix due to the ruthenium intercalation. [ABSTRACT FROM AUTHOR]