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At least 271 records · Page 15Linked to original sources
PubMed1999

Left-handed Z-DNA: structure and function.

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

Adenosine Deaminase↗
PubMed1989

Probing of DNA structure with osmium tetroxide. Effect of ligands.

Fourteen OsO4 complexes with different ligands were tested as probes of DNA structure. Of these complexes, only OsO4-2,2'-bipyridine (Os-bipy), OsO4-bathophenanthrolinedisulfonic acid (Os-bpds) and OsO4-N,N,N',N'-tetramethylenediamine (Os-TMEN) site-specifically modified the ColE1 cruciform in a supercoiled plasmid pColIR215 at millimolar concentrations. Os-bipy, Os-bpds and Os-TMEN also displayed site-specific modification of the B-Z junctions in the supercoiled plasmid pRW751 containing (dC-dG)n inserts.

Bacteriocin Plasmids↗
PubMed1989

Prediction of DNA structure from sequence: a build-up technique.

A build-up technique has been devised that permits prediction of DNA structure from sequence. No experimental information is employed other than the force field parameters. This strategy for dealing with the multiple minimum problem requires a supercomputer to make the necessary global searches. The number of energy minimization trials that were made for each of the 16 deoxydinucleoside monophosphate conformational building blocks of DNA was 1944. As a test case, the minimum energy conformations of d(GpC) and d(CpG) to 5.5 kcal/mole were then combined to generate energy-minimized structures for d(CpGpC). The number of trials that were made for d(CpGpC) was 3752. Minima for this single-stranded trimer to 15 kcal/mole were then employed to search for minimum energy conformations of the duplex d(CpGpC).d(GpCpG). The number of starting conformations that were utilized at this stage was 1514. The lowest energy duplex had a Z-II-DNA conformation, followed by a B-DNA form at 1.2 kcal/mole. The A- and Z-I-forms as well as many novel Watson-Crick base-paired structures were found at higher energy. Finally, energy-minimized structures of d(CG)6.d(CG)6 in Z-II and B-DNA conformations were computed using torsion angles from the analogous duplex trimer minima.

Base Sequence↗
PubMed2004

Models of granulocyte DNA structure are highly predictive of myelodysplastic syndrome.

We have used statistical models based on Fourier transform-infrared spectra to differentiate between the DNA structure of normal granulocytes and those obtained from patients with myelodysplastic syndrome (MDS). The substantial degree of discrimination achieved between the two DNA groups is attributed to differences in the nucleotide base and backbone structures. These structural differences allowed for the development of a discriminant analysis model that predicted, with high sensitivity and specificity, which DNA came from normal granulocytes vs. granulocytes from MDS patients. The findings are a promising basis for developing a blood test to diagnose and predict the occurrence of MDS, for which there is currently a paucity of molecular markers.

Adult↗
PubMed1996

In vitro selection of optimal AbrB-binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures.

The AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters. Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination. Using in vitro selection techniques, we have isolated optimal AbrB-binding sites from oligonucleotides containing 22 or 44 random base pairs. The best of these sites have an apparent in vitro Kd which is fivefold lower than a similar-sized DNA fragment containing the sequence corresponding to the AbrB-binding site on the spo0E gene. We tested one of the sites in vivo and found that it confers AbrB-mediated control upon a promoter not normally regulated by AbrB. In each of four separate trials, the selected sites possess motifs that converge to a simple consensus. It is argued that the nature and spacing of these motifs produce a type of three-dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant.

Amino Acid Sequence↗
PubMedDate not supplied

[Cyclic AMP content, protein kinase activity, and DNA structure in resting and proliferating peripheral blood lymphocytes and in human T-lymphoma cells].

Alterations of DNA structure, of cAMP content, of cAMP-dependent histokinases (HK) activity and cAMP-independent casein kinases (CK) activity were studied during transformation of resting cells to proliferation. These patterns were studied in human lymphocytes from peripheral blood immediately after their isolation and after cultivation within 3 days in presence of concanavalin A (ConA) or without the mitogen as well as in cultivated cells of human T-lymphoma Jurkat. Increase in content of alkaline labile sites in DNA, in activity of CK as well as distinct increase in content of cAMP were detected within the first 18 hrs of lymphocytes cultivation both in presence of ConA or without it. Early steps of cell transformation from G0 phase to G1 may be related to these alterations observed. Only slight increase in content of the alkaline labile sites in DNA and decrease in cAMP content were found in both these cell cultures. Activity of CK in the lymphocytes culture not containing ConA was decreased down to initial level, while in presence of the mitogen the enzymatic activity was increased and within 3 days it reached the level of CK activity in Jurkat cells. The rate of CK relative activity, calculated as CK/cAMP or CK/HK/cAMP ratios for each cell preparation, correlated with DNA biosynthesis rate measured by 3H-thymidine incorporation. The data obtained suggest that these patterns, used in differential diagnosis of human large intestine and gastric tumors, demonstrated also the intensity of tissue proliferation.

Casein Kinases↗
PubMed2006

General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures.

(13)C, (15)N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments.

Carbon Isotopes↗
PubMed1997

Refined structure, DNA binding studies, and dynamics of the bacteriophage Pf3 encoded single-stranded DNA binding protein.

The solution structure of the 18-kDa single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been refined using 40 ms 15N- and 13C-edited NOESY spectra and many homo- and heteronuclear J-couplings. The structures are highly precise, but some variation was found in the orientation of the beta-hairpin denoted the DNA binding wing with respect to the core of the protein. Backbone dynamics of the protein was investigated in the presence and absence of DNA by measuring the R1 and R2 relaxation rates of the 15N nuclei and the 15N-1H NOE. It was found that the DNA binding wing is much more flexible than the rest of the protein, but its mobility is largely arrested upon binding of the protein to d(A)6. This confirms earlier hypotheses on the role of this hairpin in the function of the protein, as will be discussed. Furthermore, the complete DNA binding domain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount of spin-labeled oligonucleotide. The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)6, which indicated for instance that Phe43 forms base stacking interactions with the single-stranded DNA. Finally, all results were combined to form a set of experimental restraints, which were subsequently used in restrained molecular dynamics calculations aimed at building a model for the Pf3 nucleoprotein complex. Implying in addition some similarities to the well-studied M13 complex, a plausible model could be constructed that is in accordance with the experimental data.

Crystallography, X-Ray↗
PubMed1993

Isolation and characterization of cDNA clones encoding the Drosophila homolog of the HMG-box SSRP family that recognizes specific DNA structures.

Recently an HMG-box protein denoted SSRP1, for structure-specific recognition protein 1, has been discovered which binds to specific DNA structural elements such as the bent, unwound conformations that occur upon the formation of intrastrand crosslinks by the anticancer drug cisplatin. The SSRP family includes the mouse protein T160, which recognizes recombination signal sequences. In order to delineate functional domains more clearly, a homolog of SSRP1 was cloned from Drosophila melanogaster. This homolog maps to polytene region 60A (1-4) and shares 54% identity with human SSRP1. Comparison of the predicted amino acid sequences among SSRP family members reveals 48% identity, with structural conservation in the carboxy terminus of the HMG box as well as domains of highly charged residues. Interestingly, however, the most highly conserved regions of the protein are in the less well understood amino terminus, strongly suggesting that this portion of the protein is critical for its function.

Amino Acid Sequence↗
PubMed2004

Ape1 abasic endonuclease activity is regulated by magnesium and potassium concentrations and is robust on alternative DNA structures.

Abasic lesions are common mutagenic or cytotoxic DNA damages. Ape1 is the major human apurinic/apyrimidinic (AP) endonuclease and initiates repair of abasic sites by catalyzing strand cleavage at the lesion. I show here that Ape1 single-stranded (ss) AP site incision activity prefers 0.5 mM or 2 mM MgCl(2) and low concentrations (< or =50 mM) of KCl, whereas its double-stranded (ds) activity favors 10 mM MgCl(2) and 50 mM KCl or 2 mM MgCl(2) and 200 mM KCl. Both activities favor a pH between 7.0 and 7.5, suggesting a common catalytic mechanism. In conditions designed to mimic the intracellular environment (pH 7.2; 100 mM KCl; 1 mM MgCl(2)), Ape1 ssAP site incision activity is either about fivefold more active or approximately 20-fold less efficient than its ds activity, depending on the oligonucleotide employed. Secondary structure predictions suggest a role for the DNA conformational state in determining the effectiveness of Ape1. Ape1 complex stability in the presence of EDTA (non-incising conditions) is significantly weaker for ssDNA than dsDNA, regardless of the AP substrate. Duplexes where the AP site is positioned opposite the 3' terminus of a complementary primer strand are incised with an efficiency similar (less than twofold difference) to that of the ssAP substrate alone. Moreover, Ape1 cleaved AP sites in fork-like and bubble DNA structures with an efficiency that is identical or up to sevenfold higher than ssAP-DNA. The findings here suggest that Ape1 ssAP and dsAP endonuclease activities are regulated by sequence context and the relative concentrations of certain chemical elements in vivo, and that Ape1 incision activity occurs on complex replication, recombination, and/or transcription DNA intermediates.

DNA↗
PubMed1978

A spectroscopic and electron microscopic examination of the highly condensed DNA structures formed by denaturation in Mg(ClO4)2.

1. Thermal denaturation in 1.5 M Mg(ClO4)2 of the DNA from bacteriophage lambda results in four well-separated subtransitions, as monitored by the accompanying increase in absorbance. The midpoint of the hyperchromic spectrum is significantly lowered compared to either 1.5 M MgCl2 or 3.0 M NaClO4. 2. The first two subtransitions are associated with the melting of the A . T-richest regions of the lambda DNA, as revealed by electron micrographs following fixation with formaldehyde. 3. Commencing with the third subtransition, an unusual DNA structure is observed in electron micrographs. In this structure the A . T-rich half of the molecule appears completely condensed, whereas the G . C-rich half remains native. 4. During the fourth subtransition DNA molecules condense completely and eventually aggregate to form extremely high molecular weight particles containing centers of electron density. Tendrils of DNA, primarily duplex, radiate outward from these centers. 5. The aggregation may be reversed by the removal of magnesium. The intramolecular condensation may be at least partly reversed by increasing the Mg(ClO4)2 concentrations to saturating levels.

Coliphages↗
PubMed2002

Perylene diimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase.

Four N,N'-disubstituted perylene diimides, having different side chains, have been studied for their ability in inducing G-quadruplex DNA structures. We found that electrostatic interactions between ligands side chains and DNA grooves play a main role not only in the amount of G-quadruplex formed, but also in selecting its topology. Moreover, such compounds show also a different ability to inhibit telomerase. The correlation of these findings suggests the intriguing possibility that different G-quadruplex structures could differently inhibit the enzyme.

Base Sequence↗
PubMed1994

DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay.

The alkaline comet assay is used to detect DNA single-strand breaks in individual cells embedded in agarose, lysed to denature DNA and remove proteins, and briefly exposed to an electric field to allow broken DNA to migrate. Total DNA fluorescence, measured by staining individual comets with propidium iodide, is reduced 30-40% by low doses of ionizing radiation, N-methyl-N-nitrosoguanidine (MNNG), etoposide, or hydrogen peroxide. Three possible explanations were examined: 1) these agents produce small fragments of DNA that are lost during lysis and electrophoresis, 2) the analysis of comet images is less efficient once the DNA is small enough to migrate in the electric field, and 3) DNA staining by propidium iodide is affected by changes in DNA structure caused by strand breaks and exposure to alkali. Our results indicate that the decrease in fluorescence after low doses is best explained by a change in ability of DNA, which has been denatured by alkali and subsequently renatured, to interact with fluorescent dyes. This change in fluorescence has the practical consequence of improving the ability of the alkaline comet assay to distinguish damaged from undamaged cells.

Alkalies↗
PubMed2005

The effect of temperature on DNA structural transitions under the action of Cu2+ and Ca2+ ions in aqueous solutions.

The work examines the structural transitions of DNA under the action of Cu2+ and Ca2+ ions in aqueous solution at temperatures of 29 and 45 degrees C by ir spectroscopy. Upon binding to the divalent ions studied, DNA transits into the compact state both at 29 and 45 degrees C. In the compact state DNA remains in B-form limits. The compaction process is of high positive cooperativity. As temperature increases the divalent metal ion concentration required to induce DNA compaction decreases in the case of Cu(2+)-induced compaction and increases in the case of Ca(2+)-induced compaction. It is suggested that the mechanism of the temperature effect on DNA compaction in the presence of Cu2+ ions possessing higher affinity for DNA bases differs from that of the temperature influence on Ca(2+)-induced DNA compaction. In the case of copper ions the determining factor is the increase of binding constants of the Cu2+ ions interacting with the denatured parts formed on DNA while in the case of calcium ions it is the decreased screening action of counterions upon the increase of their hydration with temperature. The efficiency of divalent metal ions studied in inducing DNA compaction depends on hydration of counterions. DNA compaction occurs in a narrow interval of Cu2+ concentrations. As the Cu2+ ion concentration increases, DNA compaction is replaced with Cu(2+)-induced DNA aggregation. At elevated temperatures Cu(2+)-induced DNA compaction could acquire a phase transition character.

Calcium↗
PubMed1999

[Stereospecific anomalies in DNA structure of Saccharomyces chromosomes IV, VII, VIII, X and XI].

Using an original computer program we analysed complete nucleotide sequences of chromosomes IV, VII, VIII, X and XI in yeast. Data about 5 largest stereospecific anomalies in each chromosome are presented together with those for 5 genes with highest CAI in each chromosome. Clusters of different stereospecific anomalies are demonstrated, including trains of not overlapping anomalies, possibly showing areas of cooperative binding of different regulatory and structural proteins to DNA (Soidla, Lukina, 1998). Together with confirming the earlier noticed connection between stereospecific anomalies and genes with high expression level (coding mostly proteins of translation machinery of cell and glycolytic enzymes), here we also noticed an obvious high incidence of transcription apparatus genes being located at (or near) largest stereospecific anomalies.

Chromosomes, Fungal↗
PubMedDate not supplied

[Some peculiarities of the kinetics of DNA structural transition after thermal jump registered by hyperchromic effect].

Structural transition of DNA is characterized by complex optical kinetic curve. The kinetic version of Felsenfeld's method is used for estimation of concentrations of "denatured" AT- and GC-base pairs in DNA molecules, and the rate constants of the accumulation of "denatured" AT as well as GC pairs have been estimated. The possible interpretation of the kinetic data obtained may be done by assumption that the fast stage of the process reflects the accumulation of the "denatured" AT pairs, which is due to the melting of AT-rich regions in the DNA molecules, whereas the slow stage of the process is limited by the melting of GC-rich regions. In contrast to the fast stage, slow stage takes place at conditions, when unwinded AT-rich regions (loops) may exist in DNA molecules and cause the delay in the process of denaturation.

Animals↗
PubMed2002

Influence of Ca2+ cations on low pH-induced DNA structural transitions.

A confocal Raman microspectrometer was used to investigate the influence of Ca2+ cations on low pH-induced DNA structural changes. The effects of Ca2+ cations on the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Based on the observation that the midpoint of the transition of Watson-Crick GC base pairs to protonated GC base pairs lies at around pH 3 (analyzing the 681 cm(-1) line), measurements were carried out on calf thymus DNA at neutral pH and pH 3 in the presence of low and high concentrations of Ca2+ cations. Raman spectra show that low concentrations of Ca2+ cations partially protect DNA against protonation of cytosine (characteristic line at 1262 cm(-1)) and do not protect adenine (characteristic line at 1304 cm(-1)) and the N(7) of guanine (line at 1488 cm(-1)) against binding of H+. High Ca2+ concentrations can prevent protonation of cytosine and protonation of adenine (little disruption of AT pairs). Analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high salt was also found to protect the N(7) of guanine against protonation.

Animals↗