Biomedical subjects

Yuri L Lyubchenko

Publications and source records attributed to Yuri L Lyubchenko.

12 recordsLinked to original sources

PubMed2003

DNA recombination: holliday junctions dynamics and branch migration.

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.

Base Sequence↗

PubMed2003

Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats.

A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.

Base Composition↗

PubMed2003

Interarm interaction of DNA cruciform forming at a short inverted repeat sequence.

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100-200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.

Animals↗

PubMed2002

Structural analysis of hemicatenated DNA loops.

BACKGROUND: We have previously isolated a stable alternative DNA structure, which was formed in vitro by reassociation of the strands of DNA fragments containing a 62 bp tract of the CA-microsatellite poly(CA).poly(TG). In the model which was proposed for this structure the double helix is folded into a loop, the base of the loop consists of a DNA junction in which one of the strands of one duplex passes between the two strands of the other duplex, forming a DNA hemicatenane in a hemiknot structure. The hemiknot DNA structures obtained with long CA/TG inserts have been imaged by AFM allowing us to directly visualize the loops. RESULTS: Here we have analyzed this structure with several different techniques: high-resolution gel electrophoresis, probing by digestion with single stranded DNA-specific nucleases or with DNase I, modification with chemicals specific for unpaired bases, and atomic force microscopy. The data show a change in DNA structure localized to the CA/TG sequence and allow us to better understand the structure of this alternative conformation and the mechanism of its formation. CONCLUSIONS: The present work is in good agreement with the model of hemicatenated DNA loop proposed previously. In the presence of protein HMGB1, shifted reassociation of the strands of DNA fragments containing a tract of the poly(CA).poly(TG) microsatellite leads to the formation of DNA loops maintained at their base by a hemicatenated junction located within the repetitive sequence. No mobility of the junction along the DNA molecule could be detected under the conditions used. The novel possibility to prepare DNA hemicatenanes should be useful to further study this alternative DNA structure and its involvement in replication or recombination.

DNA, Catenated↗

PubMed2002

Visualization of hemiknot DNA structure with an atomic force microscope.

The hemiknot, a novel type of DNA structure in which a loop is stabilized by threading one end of the duplex through another, has been studied in this paper. The hemiknot was obtained by reassociation of a DNA fragment with (CA/TG)n inserts of different lengths. Slow and fast migrating products were purified by gel electrophoresis and imaged by atomic force microscopy (AFM) using the aminopropylsilatrane-mica technique for sample preparation. Slow migrating product was characterized by the formation of small blobs for the short insert (60 bp) and clear loops and other morphologies for the long insert (188 bp). These structural features were found in almost 100% of the molecules of the slow migrating sample and were not present in the control sample. Measurements showed that the location of the blobs coincided with the positions of the inserts. The sample with the 188 bp insert in the 573 bp fragment had large structural irregularities. The majority of the molecules (77%) had asymmetrically located loops. The location of the loop in the molecules correlated well with the position of the insert in the fragment. The measured sizes of the loops were in agreement with the insert size. Altogether, these data support the hypothesis for hemiknot formation suggested earlier. In addition to looped structures, other morphologies of the hemiknot were identified in AFM images. Possible models for hemiknot formation and structure are discussed.

Animals↗

PubMed2002

Site-specific labeling of supercoiled DNA at the A+T rich sequences.

Progress in structural biology studies of supercoiled DNA and its complexes with regulatory proteins depends on the availability of reliable and routine procedures for site-specific labeling of circular molecules. For this, we made use of oligonucleotide uptake by plasmid DNA under negative superhelical tension. Subsequent circularization of the oligonucleotide label facilitated by an oligonucleotide scaffold results in its threading between the two strands of duplex DNA. Several lines of evidence, including direct AFM mapping of the label, show that the circular oligonucleotide is stably localized at its target, an A+T rich region. The specific binding mode when the oligonucleotide threads the double helix results in a DNA kink that tends to occupy an apical position in a plectonemically wound supercoiled DNA, similar to the positioning of an A-tract bend. Site-specific labels may allow visualization techniques, such as electron and atomic force microscopies, to reliably map protein binding sites, identify local alternative structures in supercoiled DNA, and monitor structural dynamics of DNA molecules in real time. Site-specific oligonucleotide reactions with DNA may also have application in biotechnology and gene therapy.

Adenine↗

PubMed2002

Atomic force microscopy study of the effects of Mg(2+) and other divalent cations on the end-to-end DNA interactions.

Atomic force microscopy (AFM) was applied to directly visualize the end-to-end DNA interaction mediated by magnesium cations. We took advantage of the APS-mica, allowing the preparation of samples in a broad range of monovalent and divalent cations to separate the effects of Mg(2+) and Na(+) cations on the interaction of restriction DNA fragments with cohesive end. The AFM data clearly show that DNA restriction fragments with cohesive ends form substantial amount of circles in the presence of Mg(2+) cations, suggesting that Mg(2+) cations stabilize the interaction of cohesive ends. This effect depends on the MgCl(2) concentration, so that the yield of circles approaches 18% in the presence of 50 mM MgCl(2). Furthermore, we demonstrate that this conferred cohesive end stability is specific for divalent cations, as substitution of MgCl(2) with NaCl leads to a near complete loss of cohesive end stability. We further demonstrate that cohesive end stabilization is achieved by substituting Mg(2+) with Ca(2+), Mn(2+), or Zn(2+). The data obtained suggest that the end stabilization mediated by divalent cations is primarily the result of inter-base interactions rather than bridging of phosphate moieties.

Cations, Divalent↗

PubMed2002

Mapping nucleosome locations on the 208-12 by AFM provides clear evidence for cooperativity in array occupation.

Concatameric sea urchin 5S rDNA templates reconstituted with histones provide very popular chromatin models for many kinds of in vitro studies. We have used AFM to characterize the locational aspects of nucleosome occupation on one such array, the 208-12, by determining the internucleosomal- and end-distance distributions for arrays reconstituted to various subsaturating levels with nonacetylated or hyperacetylated HeLa histones. A simulation analysis of the experimental distributions confirms the qualitative conclusions and provides quantitative parameter values for the identified features. For nonacetylated arrays, the end-distance data demonstrate the nucleosome positioning ability of the 5S sequence and detect an enhanced preference for nucleosomes to bind at DNA termini. The internucleosomal-distance data provide clear evidence for cooperativity in nucleosome location on these templates, detectable even at subsaturated loading levels. Hyperacetylated arrays show no change in the preference of nucleosomes to bind at termini and a slight change in nucleosome positioning behavior but, most strikingly, little or no evidence for cooperativity in nucleosome location. Thus, acetylation of the N-terminal histone tails abolishes the cooperativity.

Animals↗

PubMed2002

Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA.

Fourteen genetic neurodegenerative diseases and three fragile sites have been associated with the expansion of (CTG)n (CAG)n, (CGG)n (CCG)n, or (GAA)n (TTC)n repeat tracts. Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, slipped strand DNA, may stably and reproducibly form within triplet repeat sequences. The propensity to form slipped strand DNA is proportional to the length and homogeneity of the repeat tract. The remarkable stability of slipped strand DNA may, in part, be due to loop-loop interactions facilitated by the sequence complementarity of the loops and the dynamic structure of three-way junctions formed at the loop-outs.

DNA↗

PubMed2002

Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy.

We report here the direct evidence for the formation of alternative DNA structures in a plasmid DNA, termed pTIR10, containing a 0.23-kb pyrimidine/purine-biased (Pyr/Pur) stretch isolated from the rat genome. Long Pyr/Pur sequences are abundant in eukaryotic genomes, and they may modulate the biological activity of genes and genomes via formation of various types of triplex-related structures. The plasmid DNA in sodium acetate buffer (pH 4.35) was deposited on APS-modified mica, and after drying it was imaged with an atomic force microscope in air. Various types of thick protrusions have been observed on pTIR10 DNA. Structural parameters (width and height) of DNA molecules suggest that the alternative structures observed here are variations on the theme of an intramolecular triplex. The biological relevance of the structural features within Pyr/Pur stretches is discussed.

DNA↗

PubMedDate not supplied

Intersegmental interactions in supercoiled DNA: atomic force microscope study.

Intersegmental interactions in DNA facilitated by the neutralization of electrostatic repulsion was studied as a function of salt concentration and DNA supercoiling. DNA samples with defined superhelical densities were deposited onto aminopropyl mica at different ionic conditions and imaged in air after drying of the samples. Similar to hydrodynamic data, we did not observe a collapse of supercoiled DNA, as proposed earlier by cryo-EM studies. Instead, the formation of the contacts between DNA helices within supercoiled loops with no visible space between the duplexes was observed. The length of such close contacts increased upon increasing NaCl concentration. DNA supercoiling was a critical factor for the stabilization of intersegmental contacts. Implications of the observed effect for understanding DNA compaction in the cell and for regulation DNA transactions via interaction of distantly separated DNA regions are discussed.

DNA, Superhelical↗

PubMedDate not supplied

Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials.

The procedure of surface functionalization based on the use of 1-(3-Aminopropyl)silatrane (APS) instead of our early procedure utilizing aminopropyl triethoxy silane (APTES) is described. Unlike APTES, APS is less reactive and extremely resistant to hydrolysis and polymerization at neutral pH. The kinetics of DNA adsorption to APS-mica was studied. The results are consistent with a diffusion controlled mechanism suggesting that DNA molecules bind irreversibly with the surface upon immobilization. This conclusion is supported by the data on imaging of supercoiled DNA, the labile conformations of which are very sensitive to the conditions at the surface-liquid interface. In addition, we demonstrated directly that the segments of DNA molecules could move along the surface if the sample is imaged in aqueous solution without drying of the sample. Using the time-lapse mode of AFM imaging we visualized the transition of purine-pyrimidine sequence in supercoiled DNA from intramolecular triple-helical conformation (H-form) into the B-helix upon the change of pH from acidic (pH 5) to neutral. The mechanisms of the H-to-B transitions and the correlation of the local structural transitions with a global DNA conformation are discussed.

Adsorption↗