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At least 1,009 records · Page 56Linked to original sources
PubMed2005

Duplications between direct repeats stabilized by DNA secondary structure occur preferentially in the leading strand during DNA replication.

To ascertain a leading or lagging strand preference for duplication mutations, several short DNA sequences, i.e. mutation inserts, were designed that should demonstrate an asymmetric propensity for duplication mutations in the two complementary DNA strands during replication. The design of the mutation insert involved a 7-bp quasi inverted repeat that forms a remarkably stable hairpin in one DNA strand, but not the other. The inverted repeat is asymmetrically placed between flanking direct repeats. This sequence was cloned into a modified chloramphenicol acetyltransferase (CAT) gene containing a -1 frameshift mutation. Duplication of the mutation insert restores the reading frame of the CAT gene resulting in a chloramphenicol resistant phenotype. The mutation insert showed greater than a 200-fold preference for duplication mutations during leading strand, compared with lagging strand, replication. This result suggests that misalignment stabilized by DNA secondary structure, leading to duplication between direct repeats, occurred preferentially during leading strand synthesis.

Base Sequence↗
PubMed2000

Atomic force microscopy studies of intercalation-induced changes in plasmid DNA tertiary structure.

Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide:base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic:toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.

DNA, Superhelical↗
PubMed1991

Histone contributions to the structure of DNA in the nucleosome.

We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of approximately 10.7 base pairs per turn, while flanking regions have a periodicity of approximately 10.0 base pairs per turn. Removal of the histone tails does not change the hydroxyl radical cleavage pattern in either mixed- or unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.

Animals↗
PubMed2003

The structure of DNA in the nucleosome core.

The 1.9-A-resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein-DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fibre formation and chromatin remodelling.

Base Pairing↗
PubMed2001

A new D-DNA form of poly(dA-dT).poly(dA-dT): an A-DNA type structure with reversed Hoogsteen pairing.

The D-DNA double helix model of poly(dA-dT).poly(dA-dT) proposed in the literature is not in accordance with some notable experimental facts and physicochemical conditions to which it is related. Thus, the fibre X-ray diffraction pattern of D-DNA obtained at a relative humidity lower than that giving the A-DNA form is singularly not taken into account when one assumes that there is only one D structure of B-DNA type. We rather suggest that there are actually two different forms of D-DNA, namely D(A) which partakes in the D-A-B transitions and D(B) associated with the D-B change of conformation. Although these two DNA structures have the same helical parameters (pitch and number of residues per turn), in agreement with X-ray data, their detailed conformations are considerably different. Whereas D(B) is indeed the structure generally defined as D-DNA, a critical analysis based on a comparison between different possible DNA double helices leads us to propose dihedral angles, a set of atomic coordinates and a stereo view of another new form of D-DNA, the D(A) structural model. It is a right-handed double helix with a dinucleotide as the repeat unit. The furanose rings are of the A-DNA type (C3' endo) and the bases are hydrogen bonded according to the reversed Hoogsteen pairing. Such a disposition renders the D(A) model unsuitable for poly(dI-dC).poly(dI-dC), the other alternating polynucleotide observed in the D(B) structure. The consistency of these two different D-DNA structures of poly(dA-dT).poly(dA-dT) with the general aspects of hydration and helix-helix transitions of DNA, as well as with the conformational variability of AT base sequences, is discussed.

Base Composition↗
PubMedDate not supplied

[Relationship between tissue mitotic activity, animal age and defects in the secondary structure of DNA].

The content of defects of the secondary structure of DNA native and gamma-irradiated preparations from the intestinal mucosa and liver of adult (6-8 months) and old (24-26 months) rats was studied by the kinetic formaldehyde method. The content of the native DNA secondary structure defects does not depend on the animal and tissue mitotic activity. After gamma-irradiation in vitro (60Co, 550 R/min, 3 and 6 krad) of intestinal mucosa DNA solutions (tissues with a high mitotic activity) the content of the DNA defects is higher in old animals than in young ones. The injuring effect of gamma-rays is more pronounced in experiments with intestinal mucosa DNA than with liver DNA (tissue with a low mitotic activity). In liver DNA the content of the defects caused by gamma-rays does not depend on the animal age.

Age Factors↗
PubMed1998

Higher-order chromatin structure-dependent repair of DNA double-strand breaks: factors affecting elution of DNA from nucleoids.

The nuclear matrix is increasingly identified with the processing of DNA damage. Previous work has suggested that association of DNA with the matrix can influence the repair of DNA double-strand breaks (DSBs) and the sensitivity of mammalian cells to ionizing radiation. By selectively examining DSBs that occur as multiples (multiple DSBs) within looped DNA structures, we have identified a subset of DSBs that repair with slow kinetics through the V(D)J recombination-associated DSB repair pathway. Enrichment of S-phase populations by centrifugal elutriation and selective examination of nascent DNA by pulse-labeling were used to demonstrate that elution of DNA from nucleoids is retarded by the presence of replicating DNA. Previously, application of a Poisson-based model of induction of multiple DSBs and DNA elution to a panel of mammalian cell lines indicated that the size of the looped chromatin domains varied between cell lines. The data presented here explain the range in domain sizes between cells as the result of differences in the percentage of cells actively replicating their DNA. Correction of the model to account for S-phase populations results in a looped domain size of 2.9 Mbp independent of cell type. Single-cell gel electrophoresis of nucleoids provides additional evidence for such sized structures. Stabilization of DNA to elution during S phase does not permit repair of DSBs in the DSB repair mutants xrs5 and St.SCID, both defective for the DSB repair pathway associated with V(D)J recombination.

Animals↗
PubMedDate not supplied

[Structure of polylysine-DNA complexes].

By the methods of nuclease digestion and electron microscopy the complexes of poly-I-lysine with phage T2 DNA were studied. At micrococcal nuclease digestion of complexes polylysine--phage T2 DNA structures resistant to nuclease and containing fragments of DNA with lengths of about 300, 600, 1200 base pairs were revealed. On the electron micrographs structures superficially resembling nucleosomes were detected.

Base Composition↗
PubMed2004

Determination of the reopening temperature of a DNA hairpin structure in vitro.

A novel method, based upon primer extension, has been developed for measuring the reopening temperature of a single type of DNA hairpin structure. Two DNA oligonucleotides have been utilized and designated as primers 1 and 2. Primer 1, with its 5- and 3'-termini fully complementary to the hairpin flanking sequences, was used to evaluate primer extension conditions, and primer 2, with its 3'-end competing with the DNA hairpin stem, was used to detect the DNA hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure was predicted to prevent the annealing of the 3'-end of primer 2 with the template DNA, which leads to no primer extension. By incubating at different temperatures, the DNA hairpin structure can be reopened at a particular temperature where the primer extension can be carried out. This resulted in the appearance of double-stranded DNA that was detected on an agarose gel. This temperature is defined here as the hairpin reopening temperature.

Base Pairing↗
PubMed2005

Knowledge-based prediction of DNA atomic structure from nucleic sequence.

A simple knowledge-based method for DNA atomic structure prediction from nucleic sequence is presented. We used free B-DNA crystal structures to estimate the distribution of trinucleotide base pairs and tetranucleotide base-pair steps conformational coordinates. We used these distributions as a basis to predict the 3D position of the non-hydrogen atoms of the nucleic bases of any arbitrary DNA sequence of any length. The only constraint imposed was that the structure is a B-DNA one with Watson-Crick complementary base pairs. The method was tested on not seen DNA structures with sequence lengths varying from 6bp to 12bp. The obtained predictions have RMSE around 0.5 A for the translational conformational coordinates, and around 5 degrees for the rotational. For the estimation of the nucleic base non-hydrogen atom coordinates the RMSE is around 1.1 A. The knowledge-based method outperformed a technique based on genetic algorithms in the prediction of B-DNA structures.

Base Sequence↗
PubMed1998

Escherichia coli cAMP receptor protein-DNA complexes. 2. Structural asymmetry of DNA bending.

The effect of DNA sequence variability and the degree of cyclic AMP receptor protein (CRP)-induced bending of the flanking ends of fluorescently labeled DNA were investigated by steady-state fluorescence and differential phase polarization studies in the presence and absence of CRP. Six sequences, including the primary CRP binding sites of lac P1 (class I) and gal P1 (class II), were studied. Excitation and emission spectra of CPM-DNA upon binding CRP were observed to be qualitatively similar to one another, regardless of the CRP binding site sequence examined or the location of the probe. This result implies that the probe is not interacting with the protein. However, the magnitude of the changes in the fluorescence intensities of sensitized emission spectra of CPM-DNA is apparently dependent on the DNA sequence, indicating that the environments of the flanking ends of DNA may be different from one another in the protein-DNA complex. Differential phase polarization results were qualitatively consistent with the fluorescence energy transfer measurements. The implication of this study supports the idea that the DNA is bent symmetrically in the lac-CRP complex but is asymmetrically bent in the gal-CRP complex. The sequence in the half-site in conjunction with the flanking sequence defines the geometry of the bent DNA. It appears that the CRP-induced bend in the DNA may also be class dependent. This may be an important feature used by the system to regulate transcription at different promoter sites.

Bacterial Proteins↗
PubMed2006

Monitoring single-stranded DNA secondary structure formation by determining the topological state of DNA catenanes.

Single-stranded DNA (ssDNA) has essential biological functions during DNA replication, recombination, repair, and transcription. The structure of ssDNA must be better understood to elucidate its functions. However, the available data are too limited to give a clear picture of ssDNA due to the extremely capricious structural features of ssDNA. In this study, by forming DNA catenanes and determining their topology (the linking number, Lk) through the electrophoretic analysis, we demonstrate that the studies of catenanes formed from two ssDNA molecules can yield valuable new information about the ssDNA secondary structure. We construct catenanes out of two short (60/70 nt) ssDNA molecules by enzymatic cyclization of linear oligodeoxynucleotides. The secondary structure formed between the two DNA circles determines the topology (the Lk value) of the constructed DNA catenane. Thus, formation of the secondary structure is experimentally monitored by observing the changes of linking number with sequences and conditions. We found that the secondary structure of ssDNA is much easier to form than expected: the two strands in an internal loop in the folded ssDNA structure prefer to braid around each other rather than stay separately forming a loop, and a duplex containing only mismatched basepairs can form under physiological conditions.

DNA, Catenated↗
PubMed1992

A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome.

A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.

Agrobacterium tumefaciens↗
PubMed1996

The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA.

Large-scale deletions of mitochondrial DNA (mtDNA) are common events that have been found to occur in human ageing and in patients with mitochondrial myopathies. The mechanisms by which these deletions occur remain unclear, but several mechanisms have been proposed, such as slipped-mispairing, illegitimate recombination, and oxidative reactions elicited by free radicals. In addition, the DNA topological stress and local DNA structures have been suggested as the important factors in eliciting the recombinational events. Upon examination of 128 breakpoints of human mtDNA deletions that have been published in the past 8 years, we found that these large-scale deletions often occur at some 'hot-regions'. We thus hypothesized that there exist unusual structures in these regions of human mtDNA that are important for eliciting the deletions. To test this hypothesis, we used PCR techniques to amplify the sequences of the so-called hot-regions and analysed the PCR products by two-dimensional gel electrophoresis. We found that the sequences of nucleotide position (np) 5221-5988, np 6928-7493, np 7901-8732 and np 15327-16228 exhibited retarded mobilities like bent DNA structures; np 5989-6750, np 13282-13653 and np 13282-14850 showed increased mobilities like anti-bent DNA structures. Moreover, except for the sequences of np 1175-1766 found in 12 S and 16 S rRNA genes exhibiting abnormal mobility like bent DNA structures, we did not observe significant mobility abnormalities in the np 499-5545 region where deletions rarely occurred. We thus conclude that these hot-regions assume some kinds of unusual DNA structures, which may render these regions more sensitive to the attack of free radicals or serve as recognition motifs for certain recombination machinery that is involved in the large-scale deletions of human mtDNA.

Aging↗
PubMed1987

Heat-stable DNA polymerase I large fragment resolves hairpin structure in DNA sequencing.

A heat-stable large fragment was obtained by subtilisin digestion of DNA polymerase, prepared from Bacillus stearothermophilus. The dideoxy sequencing method, combined with the use of M13 vector has proved to be the most powerful one for obtaining the sequences of large genomes. However, the hairpin structure formed along the single-stranded DNA template often prevents the DNA polymerase from moving on, with the result that no sequence information can be obtained. The heat-stable large fragment that we have obtained has proved to be the most active at 65 degrees C. When the sequencing reaction was carried out at this temperature, the hairpin structure was resolved and the sequencing gels obtained were satisfactory.

Base Sequence↗
PubMed1996

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site.

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.

DNA↗
PubMed1985

Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA.

Two lambda phage clones carrying mitochondrial-DNA-like (mtDNA-like) sequences isolated from a human gene library were named Lm E-1 and Lm C-2, and their DNA structures were characterized. Lm E-1 contains about 0.4 kb DNA homologous to the 5' portion of the mitochondrial 16S ribosomal RNA (rRNA) gene and Lm C-2, a 1.6 kb DNA homologous to the 3' portion of the 12S rRNA gene and to almost all of the 16S rRNA gene. Comparisons of their nucleotide sequences with those of the corresponding regions of the human mtDNA revealed no detectable DNA rearrangement and their homologies to the human mtDNA are 84% and 80%, respectively. There are neither terminal repeats in the nuclear mtDNA-like sequences nor duplications of the nuclear DNAs flanking the mtDNA-like sequences. Evolutionary relationship between these two human nuclear mtDNA-like sequences and the human and bovine mtDNAs is discussed.

Animals↗