Effect of a neutralized phosphate backbone on the minor groove of B-DNA: molecular dynamics simulation studies

Alternative models have been presented to provide explanations for the sequence-dependent variation of the DNA minor groove width. In a structural model groove narrowing in A-tracts results from direct, short-range interactions among DNA bases. In an electrostatic model, the narrow minor groove of A-tracts is proposed to respond to sequence-dependent localization of water and cations. Molecular dynamics simulations on partially methylphosphonate substituted helical chains of d(TATAGGCCTATA) and d(CGCGAATTCGCG) duplexes have been carried out to help evaluate the effects of neutralizing DNA phosphate groups on the minor groove width. The results show that the time-average minor groove width of the GGCC duplex becomes significantly more narrow on neutralizing the phosphate backbone with methylphosphonates. The minor groove of the AATT sequence is normally narrow and the methylphosphonate substitutions have a smaller but measurable affect on this sequence. These results and models provide a system that can be tested by experiment and they support the hypothesis that the electrostatic environment around the minor groove affects the groove width in a sequence-dependent dynamic and time-average manner.     

Charge transport in DNA

The base pair stack within double helical DNA provides an effective medium for charge transport. The DNA pi-stack mediates oxidative DNA damage over long molecular distances in a reaction that is exquisitely sensitive to the sequence-dependent conformation and dynamics of DNA. A mixture of tunneling and hopping mechanisms have been proposed to account for this long-range chemistry, which is gated by dynamical variations within the stack. Electrochemical sensors have also been developed, based upon the sensitivity of DNA charge transport to base pair stacking, and these sensors provide a completely new approach to diagnosing single base mismatches in DNA and monitoring protein-DNA interactions electrically. DNA charge transport, furthermore, may play a role within the cell and, indeed, oxidative damage to DNA from a distance has been demonstrated in the cell nucleus. As a result, the biological consequences of and opportunities for DNA-mediated charge transport now require consideration.     

Interindividual variation in epigenomic phenomena in humans

Our knowledge of regulatory mechanisms of gene expression and other chromosomal processes related to DNA methylation and chromatin state is continuing to grow at a rapid pace. Understanding how these epigenomic phenomena vary between individuals will have an impact on understanding their broader role in determining variation in gene expression and biochemical, physiological, and behavioural phenotypes. In this review we survey recent progress in this area, focusing on data available from humans. We highlight the role of obligatory (sequence-dependent) epigenomic variation as an important mechanism for generating interindividual variation that could impact our understanding of the mechanistic basis of complex trait architecture.     

Indirect recognition in sequence-specific DNA binding by Escherichia coli integration host factor: the role of DNA deformation energy

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.     

DNA structural variations in the E. coli tyrT promoter

X-ray studies have established that the structure of a right-handed, Watson-Crick double helix can change from place to place along its length as a function of base sequence. The base pairs transmit deformations out to the phosphate backbone, where they can then be recognized by proteins and other DNA-binding reagents. Here we have examined at single-bond resolution the interactions of three commonly used nucleases (DNAase I, DNAase II, and copper-phenanthroline) with a DNA of natural origin, the 160 bp tyrT promoter. All three of these reagents seem sensitive to DNA backbone geometry rather than base sequence per se. Their sequence-dependent patterns of cleavage provide evidence for structural polymorphism of several sorts: global variation in helix groove width, global variation in radial asymmetry, and local variation in phosphate accessibility. These findings explain how sequence zones of a certain base composition, or purine-pyrimidine asymmetry, can influence the recognition of DNA by protein molecules.     

DNA shape, genetic codes, and evolution

Although the three-letter genetic code that maps nucleotide sequence to protein sequence is well known, there must exist other codes that are embedded in the human genome. Recent work points to sequence-dependent variation in DNA shape as one mechanism by which regulatory and other information could be encoded in DNA. Recent advances include the discovery of shape-dependent recognition of DNA that depends on minor groove width and electrostatics, the existence of overlapping codes in protein-coding regions of the genome, and evolutionary selection for compensatory changes in nucleotide composition that facilitate nucleosome occupancy. It is becoming clear that DNA shape is important to biological function, and therefore will be subject to evolutionary constraint.     

Sequence-dependent DNA curvature and flexibility from scanning force microscopy images

This paper reports a study of the sequence-dependent DNA curvature and flexibility based on scanning force microscopy (SFM) images. We used a palindromic dimer of a 1878-bp pBR322 fragment and collected a large pool of SFM images. The curvature of each imaged chain was measured in modulus and direction. It was found that the ensemble curvature modulus does not allow the separation of static and dynamic contributions to the curvature, whereas the curvature, when its direction in the two dimensions is taken into account, permits the direct separation of the intrinsic curvature contributions static and dynamic contributions. The palindromic symmetry also acted as an internal gauge of the validity of the SFM images statistical analysis. DNA static curvature resulted in good agreement with the predicted sequence-dependent intrinsic curvature. Furthermore, DNA sequence-dependent flexibility was found to correlate with the occurrence of A.T-rich dinucleotide steps along the chain and, in general, with the normalized basepair stacking energy distribution.     

The crystal structures of psoralen cross-linked DNAs: drug-dependent formation of Holliday junctions

The single-crystal structures are presented for two DNA sequences with the thymine bases covalently cross-linked across the complementary strands by 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). The HMT-adduct of d(CCGCTAGCGG) forms a psoralen-induced Holliday junction, showing for the first time the effect of this important class of chemotheraputics on the structure of the recombination intermediate. In contrast, HMT-d(CCGGTACCGG) forms a sequence-dependent junction. In both structures, the DNA duplex is highly distorted at the thymine base linked to the six-member pyrone ring of the drug. The psoralen cross-link defines the intramolecular interactions of the drug-induced junction, while the sequence-dependent structure is nearly identical to the native Holliday junction of d(CCGGTACCGG) alone. The two structures contrast the effects of drug- and sequence-dependent interactions on the structure of a Holliday junction, suggesting a role for psoralen in the mechanism to initiate repair of psoralen-lesions in mammalian DNA.     

Sulfur signaling: is the agent sulfide or sulfane?

Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction.     

Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.     

Curved DNA

A priori considerations and the concept of the sequence-dependent local curving of the DNA axis. Experimental evidence: electric dichroism (relaxation time measurements); anomalous electrophoretic mobility and gel-filtration of some restriction fragments of DNA; one-sided binding of the nucleosomal DNA to the mica surface. Theoretical predictions concerning the nucleotide sequences of the curved DNA. Discovery of the dinucleotide periodicity in the chromatin DNA. The sequence periodicity as a tool for mapping of the nucleosomes along the sequences. Preferential binding of the histone octamers to the curved pieces of DNA--sequence analysis predictions and comparison with experiments: Theoretical and experimental estimates of the tilt and roll angles for different combinations of the neighboring base-pairs. Inherent sequence-dependent curvature and apparent persistence length of DNA.     

Nucleotide variation and molecular structure of the heterochromatic repetitive AluI DNA in the brine shrimp Artemia franciscana

Summary It has been suggested that DNA bending could play a role in the regulation of gene expression, chromosome segregation, specific recombination and/or DNA packaging. We have previously demonstrated that an Alul DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp A. franciscana. By the analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments we verified that the repetitive AluI DNA shows a stable curvature that determines a solenoidal geometry to the double helix. This particular structure could be of relevant importance in conferring the characteristic heterochromatic condensation. In this paper we evaluate how the point mutations that occurred during the evolution of the Alul sequence of A. franciscana could influence the sequence-dependent tridimensional conformation. The obtained data underline that, in spite of the high sequence mutation frequency (10%) of the repetitive DNA, the general structure of the heterochromatic DNA is not greatly influenced, but rather there is a substantial variation of the copy number of the repetitive AluI fragment. This variation could be responsible for the hypothetical function of the constitutive heterochromatin.Offprint requests to: N. Landsberger     

Local supercoil-stabilized DNA structures

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.     

The protection role of heat shock protein 70 (HSP-70) in the testes of cadmium-exposed rats

Cadmium (Cd) is an environmental carcinogenic pollutant known to inactivate several proteins involved in DNA repair systems while at the same time creating an oxidative stress that can result in additional DNA lesions. The testis and the lung are the target organs for cadmium carcinogenesis. Increased production of oxidants in vivo can cause damage to intracellular macromolecules such as DNA, proteins and lipids, which in turn lead to oxidative injury. So, this investigation aimed to evaluate the protective role of L-Carnitine through up regulation of HSPs against DNA damage induced by cadmium chloride. The current study was carried out on forty adult male rats, each with average weight 220-250g., were divided into 4 equal groups. 1(st) group was received saline solution (0.5 ml/100 g body weight) and kept as control. 2(nd) group was received 500mg / kg body weight L-Carnitine intraperitoneally (IP). 3(rd) group was administered 1.2 mg cadmium chloride IP. 4(th) group was received both cadmium chloride and L-Carnitine simultaneously. The comet assay parameters showed significantly increased HSP70 and DNA damage in testis cells after 10 and 56 days in the third group. Meanwhile, HSP70 showed significantly decreased levels after 10 days and 56 days in the fourth group after L-Carnitine treatment simultaneously with cadmium chloride. The results of the present study demonstrate a damaging effect of cadmium chloride on DNA of the testis cells (with low stress response). This damaging effect increases the synthesis of HSP70 that upregulated by L-Carnitine treatment and showed ameliorative effect of the cells for recovery.     

Signatures of Protein-DNA Recognition in Free DNA Binding Sites

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.     

DNA sequence-dependent deformability--insights from computer simulations

The article reviews some recent developments in studying DNA sequence-dependent deformability, with emphasis on computer modeling. After a brief outline of available experimental techniques, we proceed to computational methods and focus on atomic-resolution molecular dynamics (MD) simulations. A sequence-dependent local (base-pair step) force field inferred from MD is compared with force fields obtained by other techniques. Various methods for establishing global (flexible-rod) DNA elastic constants are reviewed, including an approach based on atomic resolution MD. The problem of defining the global deformation variables, as well as the question of anisotropy and nonlocal effects, are discussed. As an example, both local and global deformability calculations from atomic-resolution MD of EcoRI dodecamer are presented.     

Modeling translocation dynamics of strand displacement DNA synthesis by DNA polymerase I

A model is presented for the translocation dynamics of the strand displacement DNA synthesis by DNA polymerases such as polymerase I family. (i) The model gives an explanation to the experimental results which showed that the rate of strand displacement DNA synthesis is nearly consistent with that of single stranded primer extension synthesis, although the two are expected to have substantial differences in their energetics. (ii) During strand displacement DNA synthesis, the pausing at the specific sequence is considered to be due to an affinity of the fingers subdomain for the specific sequence of dsDNA downstream of the single strand. The theoretical results on the sequence-dependent pausing dynamics such as the mean pausing lifetimes and the distribution of the pausing lifetime are consistent with the experimental data. Moreover, predicted results are presented for the binding affinity of the fingers subdomain for the specific sequence of dsDNA and the dependence of the mean sequence-dependent pausing lifetime on the external force acting on the polymerase.     

Sequence-specific damage induced by the impact of 3-30 eV electrons on oligonucleotides.

The ability of low-energy electrons to induce single- and double-strand breaks in DNA has recently been demonstrated. Here we show the propensity of 3-30 eV electrons to initiate base sequence-dependent damage to a short single DNA strand. Solid monolayer films of homogeneous thymidine (T(9)) and deoxycytidine (dCy(9)) and heterogeneous oligomers (T(6)dCy(3)) are bombarded with 1-30 eV electrons in an ultrahigh-vacuum system. CN, OCN and/or H(2)NCN are detected by a mass spectrometer as the most intense neutral fragments desorbing in vacuum. A weaker signal of CH(3)CCO is also detected, but only from oligonucleotides containing thymine. Below 17 eV, the energy dependence of the yields of CN, OCN and CH(3)CCO exhibits resonance-like structures, attributed to dissociative electron attachment (DEA). Above 17 eV, the monotonic increase in the fragment yields indicates that nonresonant processes (i.e. dipolar dissociation) control the fragmentation of these molecules. Within the energy range investigated, comparison of the magnitude of the total fragment yields produced by electron attack on dCy(9), T(6)-dCy(3) and T(9) suggests the following order in the sensitivity of single-strand DNA: dCy(9) > T(6)-dCy(3) > T(9). At 12 eV, the total fragment yields are found to be 5.8, 5.0 and 3.9 x 10(-3) fragment/electron, respectively. From the yields obtained with the two homo-oligonucleotides, we differentiate between contributions arising from the chemical nature of the base and the effect of environment (i.e. the sequence) when a thymidine unit in T(9) is replaced by dCy. The base sequence-dependent damage is found to vary with incident electron energy. These results reinforce the idea that genomic sensitivity to ionizing radiation depends on local genetic information. Furthermore, they underscore the possible role of low-energy electrons in the pathways responsible for the induction of specific genomic lesions.