Alterations In DNA Conformation Research Articles

Dessecting the toxicological profile of polysorbate 80 (PS80): comparative analysis of constituent variability and biological impact using a zebrafish model

Polysorbate 80, commonly abbreviated as PS80, is a widely used pharmaceutical excipient renowned for its role as a solubilizer and stabilizer in drug formulations. Although PS80 is essential for various pharmaceutical applications, particularly in the formulation of injectable drugs, it has been implicated in a range of adverse reactions. However, due to the complexity of the composition of PS80, the differences in the types and contents of the constituents of PS80 from different manufacturers increase the probability or likelihood of their uneven quality.Addressing the complete spectrum of PS80′s components is challenging; thus, most studies to date have examined PS80 as a singular entity. This approach, however, carries a degree of uncertainty, as it overlooks the unique composition and concentration of components within the PS80 used in experiments, which may not reflect the actual diversity in commercially available PS80 products.Recognizing the critical need to understand how PS80′s composition influences biological effects and toxicity, our study aims to bridge this knowledge gap. By doing so, we can clarify how different PS80 compositions from various manufacturers might affect the quality of pharmaceutical formulations, and also guide excipient manufacturers toward producing higher-quality PS80. Such insights could further facilitate a more targeted application of PS80 in drug development.Building on our previous work, we isolated and prepared two key components of PS80—polyoxyethylene sorbitan monooleate (PSM) and polyoxyethylene isosorbide monooleate (PIM)—and conducted a systematic comparison. We evaluated the acute, hemolytic, and target organ toxicity of two different PS80 samples, as well as PSM and PIM, using a zebrafish model. Our research also delved into the potential mechanisms behind the observed toxicological effects, providing an in-depth understanding of PS80′s impact on biological systems.The results show that PS80, PSM, and PIM resulted in developmental anomalies in larval zebrafish. The primary organs of acute toxicity in zebrafish exposed to PS80 and its typical components PSM and PIM include the cardiovascular system, kidneys, intestines, skin, and liver. Notably, PIM further induced severe pericardial edema and erythrocyte hemolysis, thereby affecting blood flow. The samples also instigated oxidative damage by disrupting the redox equilibrium in the larvae. Compared to PS80, both PSM and PIM induced greater oxidative damage, with PIM notably causing significantly higher lipid oxidation, suggesting that oxidative stress may play a crucial role in polysorbate80-induced toxicity. Furthermore, our study found that PS80 could induce alterations in DNA conformation. The findings underscore the necessity for excipient regulators to establish comprehensive quality standards for Polysorbate 80 (PS80). By implementing such standards, it is possible to minimize the clinical risks associated with the variability in PS80 composition, ensuring safer pharmaceutical products for patients.

Fructose derived oligosaccharides prevent lipid membrane destabilization and DNA conformational alterations during vacuum-drying of Lactobacillus delbrueckii subsp. bulgaricus

Dehydration of lactic acid bacteria for technological purposes conducts to multilevel damage of bacterial cells. The goal of this work was to determine at which molecular level fructose-oligosaccharides (FOS) and sucrose protect Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 during the vacuum-drying process. To achieve this aim, the cultivability and metabolic activity of vacuum-dried bacteria were firstly determined (plate counting and absorbance kinetics). Then, the membrane integrity and fluidity were assessed using propidium iodide and Laurdan probes (general polarization -GP-), respectively. Finally, bacterial structural alterations were determined using high throughput methods (fluorescence confocal microscopy and Raman spectroscopy coupled to Multivariate Curve Resolution analysis -MCR-). The vacuum-drying process directly affected the microorganism's cultivability and membrane integrity. Non-dehydrated cells and sugar protected bacteria (both with FOS or sucrose) presented high GP values typical from the gel state, as well as phospholipids microdomains laterally organized along the cytoplasmic membrane. On the contrary, bacteria dehydrated without protectants presented low GP values and greater water penetration, associated with membrane destabilization. Raman spectroscopy of vacuum-dried cells revealed DNA conformational changes, B-DNA conformations being associated to non-dehydrated or sugar protected bacteria, and A-DNA conformations being higher in bacteria vacuum-dried without protectants. These results support the role of FOS and sucrose as protective compounds, not only acting at the membrane organizational level but also preventing conformational alterations of intracellular structures, like DNA.

Punicalagin from pomegranate promotes human papillary thyroid carcinoma BCPAP cell death by triggering ATM-mediated DNA damage response

Punicalagin (PUN), a component derived from pomegranate, is well known for its anticancer activity. Our previous work revealed that PUN induces autophagic cell death in papillary thyroid carcinoma cells. We hypothesized that PUN triggers DNA damage associated with cell death because DNA damage was reported as an inducer of autophagy. Our results showed that PUN treatment caused DNA breaks as evidenced by the significant enhancement in the phosphorylation of H2A.X. However, reactive oxygen species and DNA conformational alteration, 2 common inducing factors in DNA damage, were not involved in PUN-induced DNA damage. The phosphorylation of ataxia-telangiectasia mutated gene-encoded protein (ATM) but not ataxia telangiectasia and Rad3-related protein (ATR) was up-regulated in a time- and dosage-dependent manner after PUN treatment. KU-55933, an inhibitor of ATM, inhibited the phosphorylation of ATM induced by PUN and reversed the decreased cell viability caused by PUN. Thus, we demonstrated that PUN induces cell death of papillary thyroid carcinoma cells by triggering ATM-mediated DNA damage response, which provided novel mechanisms and potential targets for the better understanding of the anticancer actions of PUN.

Plasmonic Circular Dichroism Study of Gold Nanorod-Quadruplex Nanobioconjugates

Circular dichroism spectroscopy is a simple way to study G-quadruplex structure and is very useful for monitoring the conformational changes in G-quadruplex structure induced by modifications of the environment. Plasmonic nanoparticles with localized surface plasmon resonance (LSPR) can create strong electromagnetic fields at the surface of plasmonic metals, which remarkably influence the optical properties of molecules. Plasmonic CD is a new CD signal which originates from the dipole–dipole interactions between surface plasmons of NPs and chiral biomolecules have received interest research in various fields of nanotechnology. In this paper we study the interaction between gold nanorods (GNR) and two types of G-quadruplex (parallel and antiparallel), to monitor the alterations in DNA conformation and plasmonic CD signal upon formation of GNR-quadruplex nanobioconjugate. The results from this study indicate the plasmonic CD signals in visible regions are more sensitive than Far-UV CD signals to detect the spatial conformational state of G-quadruplex-GNR nanobioconjugate.

Accelerated processing of solitary and clustered abasic site DNA damage lesions by APE1 in the presence of aqueous extract of Ganoderma lucidum.

The stimulatory effect of the aqueous extract of G. lucidum, a basidiomycetes class fungus in the APE1-enzyme-mediated processing of solitary and bistranded clustered abasic sites DNA damages is presented. Abasic sites are considered the most common type of DNA damage lesions. Our study shows enhanced activity of APE1 in the processing of abasic sites in the presence of the polysaccharides fraction of G. lucidum. Remarkable increase in the amount of single-strand breaks (SSBs) and double-strand breaks (DSBs) from solitary and bistranded clustered abasic sites respectively with APE1 in the presence of the extract was found. This trend is maintained when abasic sites in DNA oligomers are exposed to fibroblast cell extracts in the presence of the extract. While DNA conformational alteration is negligible, APE1 enzyme shows characteristic changes in the alpha helix and beta strand ratio after incubation with G. lucidum extract. The enhanced reactivity of APE1 at the molecular level in the presence of G. lucidium is attributed to this effect. This study potentially amplifies the scope of the use of G. lucidum, which was earlier shown to have only reactive oxygen species (ROS) scavenging properties with regards to DNA damage inhibition.

Isolating stem cells in the inter-follicular epidermis employing synchrotron radiation-based Fourier-transform infrared microspectroscopy and focal plane array imaging

Normal function and physiology of the epidermis is maintained by the regenerative capacity of this tissue via adult stem cells (SCs). However, definitive identifying markers for SCs remain elusive. Infrared (IR) spectroscopy exploits the ability of cellular biomolecules to absorb in the mid-IR region (λ = 2.5-25 μm), detecting vibrational transitions of chemical bonds. In this study, we exploited the cell's inherent biochemical composition to discriminate SCs of the inter-follicular skin epidermis based on IR-derived markers. Paraffin-embedded samples of human scalp skin (n = 4) were obtained, and 10-μm thick sections were mounted for IR spectroscopy. Samples were interrogated in transmission mode using synchrotron radiation-based Fourier-transform IR (FTIR) microspectroscopy (15 × 15 μm) and also imaged employing globar-source FTIR focal plane array (FPA) imaging (5.4 × 5.4 μm). Dependent on the location of derived spectra, wavenumber-absorbance/intensity relationships were examined using unsupervised principal component analysis. This approach showed clear separation and spectral differences dependent on cell type. Spectral biomarkers concurrently associated with segregation of SCs, transit-amplifying cells and terminally-differentiated cells of epidermis were primarily PO(2)(-) vibrational modes (1,225 and 1,080 cm(-1)), related to DNA conformational alterations. FPA imaging coupled with hierarchical cluster analysis also indicated the presence of specific basal layer cells potentially originating from the follicular bulge, suggested by co-clustering of spectra. This study highlights PO (2) (-) vibrational modes as potential putative SC markers.

Investigation of DNA Spectral Conformational Changes and Polymer Buffering Capacity in Relation to Transfection Efficiency of DNA/Polymer Complexes

The relation between transfection efficiency of DNA/polymer complexes and DNA conformational alterations is investigated. The buffering capacity of several synthetic polymers is also studied to relate their performance in transfection efficiency. The cationic polymer/DNA interaction was evaluated by measuring the alteration of DNA secondary structures in solution before and after the addition of polymer with ATR-FTIR technique. The degree of protonation in aqueous cationic polymers is varied upon pH and different structures. A polymer capable of protonation acts like a proton sponge to react with H+ in titration with HCl. This characteristic was evaluated in relation to transfection efficiency because the capacity would help the release of endocytotic DNA from endosome/lysosome on its way to expression. IR results show that the antisymmetric PO2- vibration of DNA (at 1224 cm-1) shifts toward lower frequencies in complexation with PEI or PLLys (these polymers are able to transfect DNA). By contrast, the antisymmetric PO2- vibration of DNA in presence of PDAMA or dextran (these polymers are poor in DNA transfection) shows a shifting to higher frequencies or no alteration was observed. Interestingly, the polymers with best performance in transfection efficiency are in this order: PEI>PDMAEMA>PLLys>PDAMA>dextran which is in the same order as their polymer buffering capacity. These facts indicate polymers possessing better buffering capacity could result in higher transfection efficiency. Also, we have demonstrated in this paper that the alteration in the antisymmetric PO(2)(-) stretching vibration in the IR spectra is sensitive to the binding of cationic polymers with DNA and the transfection efficiency of the formed complexes. These findings are useful for the developing polymer-based gene delivery systems with better in vitro and in vivo performance. NOVELTY OF THE WORK: this work establishes a rational relation between transfection efficiency of DNA/polymer complexes and DNA conformational alterations. Also, the buffering capacity of polymers is found to be closely related to their performance in transfection efficiency.

Fourier Transform Infrared Microspectroscopy Identifies Symmetric PO2− Modifications as a Marker of the Putative Stem Cell Region of Human Intestinal Crypts

Abstract Complex biomolecules absorb in the mid-infrared (λ = 2–20 μm), giving vibrational spectra associated with structure and function. We used Fourier transform infrared (FTIR) microspectroscopy to “fingerprint” locations along the length of human small and large intestinal crypts. Paraffin-embedded slices of normal human gut were sectioned (10 μm thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected using globar (15 μm × 15 μm aperture) FTIR microspectroscopy in reflection mode, synchrotron (≤10 μm × 10 μm aperture) FTIR microspectroscopy in transmission mode or near-field photothermal microspectroscopy. Dependent on the location of crypt interrogation, clear differences in spectral characteristics were noted. Epithelial-cell IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in “hyperspace” might on the basis of multivariate distance reveal biophysical differences along the length of gut crypts. Following spectroscopic analysis, plotted clusters and their loadings plots pointed toward symmetric (νs)PO2− (1,080 cm−1) vibrations as a discriminating factor for the putative stem cell region; this proved to be a more robust marker than other phenotypic markers, such as β-catenin or CD133. This pattern was subsequently confirmed by image mapping and points to a novel approach of nondestructively identifying a tissue's stem cell location. νsPO2−, probably associated with DNA conformational alterations, might facilitate a means of identifying stem cells, which may have utility in other tissues where the location of stem cells is unclear. Disclosure of potential conflicts of interest is found at the end of this article.

Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes

Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.

Base Flipping in Nucleotide Excision Repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.

First evidence for helical transitions in supercoiled DNA by amyloid Beta Peptide (1-42) and aluminum: a new insight in understanding Alzheimer's disease.

Previously, we evidenced a B --> Z helical change in Alzheimer's brain genomic DNA, leading to a hypothesis that Alzheimer's disease (AD) etiological factors such as aluminum (Al), amyloid beta (Abeta) peptide, and Tau might play a role in modulating DNA topology. In the present study, we investigated the interaction of Al and Abeta with DNA. Our results show that Abeta(1-42) could induce a B --> Psi (Psi) conformational change in pUC 18 supercoiled DNA (scDNA), Abeta(1-16) caused an altered B-form, whereas Al induced a complex B-C-A mixed conformation. Ethidium bromide binding and agarose gel electrophoresis studies revealed that Al uncoiled the DNAto a fully relaxed form, whereas Abeta(1-42) and Abeta(1-16) effected a partial uncoiling and also showed differential sensitivity toward chloroquine-induced topoisomer separation. Our findings show for the first time that Abeta and Al modulate both helicity and superhelicity in scDNA. A new hypothetical model explaining the potential toxicity of Abeta and Al in terms of their DNA binding properties leading to DNA conformational alteration is proposed.

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5′-CAGCAG-3′ transferring a methyl group from S-adenosyl- l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.

Yeast Rad54 Promotes Rad51-dependent Homologous DNA Pairing via ATP Hydrolysis-driven Change in DNA Double Helix Conformation

Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA.

Sequence-specific DNA Binding of the Phage Mu C Protein : Footprinting Analysis Reveals Altered DNA Conformation Upon Protein Binding

The momgene of bacteriophage Mu, which codes for a DNA modification function, is regulated in a complex manner at both transcriptional and translational levels. The phage-encoded C protein functions as an activator of momtranscription. The mompromoter has features of an activator-dependent weak promoter, and the C binding site is located upstream and overlapping the −35 region and includes the palindromic sequence TTAT(N) 6ATAA. The interactions of this activator protein at its binding site in P mom has been investigated using four different chemical footprinting reagents. The protein footprint spans a region of 18 to 25 bp, depending on the nature of the chemical reagent used. Dimethylsulfate protection experiments revealed the base-specific interactions. The protected guanines are separated by 15 bp and are located beyond the interrupted palindromic sequence. A tripartite footprint was observed with hydroxyl radical, generated by Fe(II)-EDTA, which shows the binding of the protein to one face of the helix. The extent of protection conferred by the bound protein, however, is not uniform, suggesting that the interaction is asymmetric. The chemical nuclease 1,10-phenanthroline-copper, a minor groove specific ligand, shows hyper-reactivity upon protein binding in the top strand nucleotide triplet CAC, again confirming the protein-induced alterations in DNA conformation. Gel exclusion chromatography and chemical crosslinking experiment with the purified protein suggest that this mode of interaction is accomplished by a dimeric protein. This observation is supported by electrophoretic mobility shift assay using heterodimer of pure C protein and staphylococcal protein A-C fusion. The deletion analysis implicates a role for the carboxyl-terminal region of the protein in DNA binding.

Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo.

Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsional strain might assist strand separation and facilitate factor binding. Therefore, the interaction of FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K with supercoiled DNA was studied. Remarkably, both proteins recognize their respective elements torsionally strained but not as liner duplexes. Single-strand- or supercoil-dependent gene regulatory proteins may directly link alterations in DNA conformation and topology with changes in gene expression.

Targeted base stacking disruption by the EcoRI DNA methyltransferase.

We describe a novel fluorescence-based assay for detecting DNA conformational alterations within enzyme-DNA complexes. The target adenine for EcoRI DNA methyltransferase (GAATTC) was replaced with 2-aminopurine, which fluoresces upon excitation at 310 nm. Addition of the methyltransferase to the duplex binding site results in a 14-fold increase in fluorescence intensity with a 10 nm blue shift. The fluorescence is approximately 50% of that observed with equimolar free nucleoside, consistent with extrahelical stabilization of the target base in the enzyme-DNA complex. The shift in lambda max further implies the base is placed into a low dielectric environment. For adenine-specific DNA methyltransferases, a hydrophobic pocket composed of highly conserved amino acids lies proximal to the cofactor binding site. Substitution of 2-aminopurine adjacent to the target base also results in detectable changes in fluorescence emission following complex formation with the methyltransferase. Thus, other classes of enzymes hypothesized to utilize base flipping can be investigated by this method.

Platinum(II) complexes containing iminoethers: a trans platinum antitumour agent

The biological activity of cm and trans complexes of formula [PtCl 2[HNC(OMe)Me] 2] has been investigated. The iminoether ligands can have either E or Z configuration about the C N double bond, therefore EE, EZ and ZZ isomers are obtainable. Substitution of iminoether with EE configuration for amine leads to unexpectedly high antitumour activity for the complex with trans geometry which turns out to be more active than the cis congener in the P388 leukaemia system. The same trans-EE complex shows an activity comparable to that of cisplatin in reducing the primary tumour mass and lung metastases in mice bearing Lewis lung carcinoma, thus representing a trans platinum complex active on both limphoproliferative and solid metastasizing murine tumours. Also the cytotoxicity, the inhibition of DNA synthesis and the mutagenic activity, which are greater for the cis- with respect to the trans-isomer in the amine complexes, are instead greater for the trans- than for the cis-isomer in the case of iminoether compounds. Binding to calf thymus DNA is slower for iminoether complexes than it is for amine complexes, however after 24 h reaction time the level of binding is similar for both types of complexes. Trans-EE, like trans-DDP, does not give the DNA conformational alterations (terbium fluorescence) typical of antitumour-active cis-platinum compounds, but, under strictly analogous experimental conditions, shows a greatly reduced DNA interstrand cross-linking ability (heat denaturation/renaturation assay) with respect to either trans-DDP or cis-EE and cis-DDP. The data in hand point to a new trans platinum antitumour complex with a mechanism of action different from that of cis-DDP and classical analogues.

Editorial overview

Chemical probes sensitive to alterations in DNA conformation, especially Z-DNA, have been identified. These permit cleavage of DNA at sites of unusual structure, the results of which can be displayed on a sequencing gel. Using supercoiled plasmids containing Inserts of d(C-G)16 and d(C-A)31·d(T-G)31, it was found that hydroxylamine and osmium tetraoxide react preferentially with cytosines and thymines, respectively, near B-DNA-Z-DNA junctions; diethylpyrocarbonate reacts more strongly with purines within Z-DNA regions; and dimethylsulfate and diethylsulfate react more strongly with guanines in Z-DNA that are out of phase with the usual pattern of purine-pyrimidine alternation. Our results show that B-Z boundaries are mobile and that with increasing torsional strain, the Z-DNA regions can expand to include nonalternating nucleotide sequences.

Altered electrophoretic behavior of DNA due to short-time UV-B irradiation.

UV-B irradiation is often inevitable for visualization of DNA fragments after ethidium bromide staining. Three different simple-repeat-containing, double-stranded genomic DNA fragments were analyzed for UV-B (312 nm) damage using different gel electrophoretic systems. The effects of UV-B light were obvious after 5 min (31.5 kJ/m2) of irradiation in native polyacrylamide gel electrophoresis (PAGE). Standard single-strand conformation analyses revealed no alterations while a modification did. Sodium dodecyl sulfate (SDS)-PAGE was found to be highly sensitive with regard to the detection of damages and their time/dosage dependency. In addition, SDS-PAGE analysis pointed to different events occurring during UV-B irradiation. Alterations in DNA conformation were detected in every single strand analyzed after 1 min (6.3 kJ/m2) of UV-B exposure. Gel retardation analyses revealed significant changes of protein binding to target DNAs after 2 min of irradiation--possibly stemming from structural modifications and/or originating from binding sites for proteins involved in DNA repair.

A DNA conformational alteration induced by a neighboring oligopurine tract on GAATTA enables nicking by EcoRI.

The pseudo EcoRI site GAATTA in the U3 region of the long terminal repeat of human immunodeficiency virus, which is flanked by a 26-base pair oligopurine tract, is readily nicked by either EcoRI or RsrI. The strand-specific nick occurs predominantly between the G and A residues and is independent of negative supercoiling. Other GAATTA sites surrounded by random (non-oligopurine) sequences are not nicked by these restriction endonucleases. However, other types and lengths of oligopurine tracts are effective in inducing the nicking in neighboring GAATTA sites. Hence, we propose that the flanking oligopurine tracts induce an altered DNA conformation on the GAATTA target site which may be similar to the transition state induced by EcoRI when binding to its canonical recognition site. Gel retardation analyses on restriction fragments containing the oligopurine-GAATTA-oligopurine sequences suggest the presence of helical axis distortions which are consistent with this interpretation.