Biomedical subjects

Vladimir N Potaman

Publications and source records attributed to Vladimir N Potaman.

13 recordsLinked to original sources

PubMed2006

Site-specific labeling of supercoiled DNA.

Visualization of site-specific labels in long linear or circular DNA allows unambiguous identification of various local DNA structures. Here we describe a novel and efficient approach to site-specific DNA labeling. The restriction enzyme SfiI binds to DNA but leaves it intact in the presence of calcium and therefore may serve as a protein label of 13 bp recognition sites. Since SfiI requires simultaneous interaction with two DNA recognition sites for stable binding, this requirement is satisfied by providing an isolated recognition site in the DNA target and an additional short DNA duplex also containing the recognition site. The SfiI/DNA complexes were visualized with AFM and the specificity of the labeling was confirmed by the length measurements. Using this approach, two sites in plasmid DNA were labeled in the presence of a large excess of the helper duplex to compete with the formation of looped structures of the intramolecular synaptic complex. We show that the labeling procedure does not interfere with the superhelical tension-driven formation of alternative DNA structures such as cruciforms. The complex is relatively stable at low and high pH (pH 5 and 9) making the developed approach attractive for use at conditions requiring the pH change.

Binding Sites↗

PubMed2006

DNA strand arrangement within the SfiI-DNA complex: atomic force microscopy analysis.

The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before cleavage of the four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving conditions (Ca2+ instead of Mg2+ in the reaction buffer) was performed. Intramolecular complexes formed by protein interaction between two binding sites in one DNA molecule (cis interaction) as well as complexes formed by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex, the angles between the DNA helices in the proximity of the complex were systematically analyzed. All the data show clear-cut bimodal distributions centered around peak values corresponding to 60 degrees and 120 degrees. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA molecules with different arm lengths flanking the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles of 60 degrees or 120 degrees between the DNA helices correspond to a complex in which one of the helices is flipped with respect to the orientation of the other. Complexes formed by five different recognition sequences (5'-GGCCNNNNNGGCC-3'), with different central base pairs, were also analyzed. Our results showed that complexes containing the two possible orientations of the helices were formed almost equally. This suggests no preferential orientation of the DNA cognate site within the complex, suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein.

Base Sequence↗

PubMed2005

Specific binding of poly(ADP-ribose) polymerase-1 to cruciform hairpins.

Poly(ADP-ribose) polymerase-1 (PARP-1) participates in DNA cleavage and rejoining-dependent reactions, such as DNA replication, recombination and repair. PARP-1 is also important in transcriptional regulation, although the determinants for its binding to undamaged genomic DNA have not been defined. Previously, we have shown by low-resolution mapping that PARP-1 may bind to the cruciform-forming regions of its own promoter. Here, using DNase I and nuclease P(1) footprinting and atomic force microscopy, we show that PARP-1 binds to stem/loop boundaries of cruciform hairpins. Cleavage of the cruciform by the junction resolvase T4 endonuclease VII is independent of PARP-1, which indicates that PARP-1 does not bind to the four-arm junctions of the cruciform. Thus, PARP-1 differs from other cruciform-binding proteins by binding to hairpin tips rather than to junctions. Furthermore, our data indicate that PARP-1 can interact with the gene regulatory sequences by binding to the promoter-localized cruciforms.

DNA↗

PubMed2005

Regulation of poly(ADP-ribose) polymerase-1 by DNA structure-specific binding.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an intracellular sensor of DNA strand breaks and plays a critical role in cellular responses to DNA damage. In normally functioning cells, PARP-1 enzymatic activity has been linked to the alterations in chromatin structure associated with gene expression. However, the molecular determinants for PARP-1 recruitment to specific sites in chromatin in the absence of DNA strand breaks remain obscure. Using gel shift and enzymatic footprinting assays and atomic force microscopy, we show that PARP-1 recognizes distortions in the DNA helical backbone and that it binds to three- and four-way junctions as well as to stably unpaired regions in double-stranded DNA. PARP-1 interactions with non-B DNA structures are functional and lead to its catalytic activation. DNA hairpins, cruciforms, and stably unpaired regions are all effective co-activators of PARP-1 auto-modification and poly(ADP-ribosyl)ation of histone H1 in the absence of free DNA ends. Enzyme kinetic analyses revealed that the structural features of non-B form DNA co-factors are important for PARP-1 catalysis activated by undamaged DNA. K0.5 constants for DNA co-factors, which are structurally different in the degree of base pairing and spatial DNA organization, follow the order: cruciform<or=hairpin<<loop. DNA structure also influenced the reaction rate; when a hairpin was substituted with a stably unpaired region, the maximum reaction velocity decreased almost 2-fold. These data suggest a link between PARP-1 binding to non-B DNA structures in genome and its function in the dynamics of local modulation of chromatin structure in the normal physiology of the cell.

Binding Sites↗

PubMed2004

Interaction of the Zalpha domain of human ADAR1 with a negatively supercoiled plasmid visualized by atomic force microscopy.

Interest to the left-handed DNA conformation has been recently boosted by the findings that a number of proteins contain the Zalpha domain, which has been shown to specifically recognize Z-DNA. The biological function of Zalpha is presently unknown, but it has been suggested that it may specifically direct protein regions of Z-DNA induced by negative supercoiling in actively transcribing genes. Many studies, including a crystal structure in complex with Z-DNA, have focused on the human ADAR1 Zalpha domain in isolation. We have hypothesized that the recognition of a Z-DNA sequence by the Zalpha(ADAR1) domain is context specific, occurring under energetic conditions, which favor Z-DNA formation. To test this hypothesis, we have applied atomic force microscopy to image Zalpha(ADAR1) complexed with supercoiled plasmid DNAs. We have demonstrated that the Zalpha(ADAR1) binds specifically to Z-DNA and preferentially to d(CG)(n) inserts, which require less energy for Z-DNA induction compared to other sequences. A notable finding is that site-specific Zalpha binding to d(GC)(13) or d(GC)(2)C(GC)(10) inserts is observed when DNA supercoiling is insufficient to induce Z-DNA formation. These results indicate that Zalpha(ADAR1) binding facilities the B-to-Z transition and provides additional support to the model that Z-DNA binding proteins may regulate biological processes through structure-specific recognition.

Adenosine Deaminase↗

PubMed2004

Supercoiling-induced DNA bending.

Local DNA bending is a critical factor for numerous DNA functions including recognition of DNA by sequence-specific regulatory binding proteins. Negative DNA supercoiling increases both local and global DNA dynamics, and this dynamic flexibility can facilitate the formation of DNA-protein complexes. We have recently shown that apexes of supercoiled DNA molecules are sites that can promote the formation of an alternative DNA structure, a cruciform, suggesting that these positions in supercoiled DNA are under additional stress and perhaps have a distorted DNA geometry. To test this hypothesis, we used atomic force microscopy to directly measure the curvature of apical positions in supercoiled DNA. The measurements were performed for an inherently curved sequence formed by phased A tracts and a region of mixed sequence DNA. For this, we used plasmids in which an inverted repeat and A tract were placed at precise locations relative to each other. Under specific conditions, the inverted repeat formed a cruciform that was used as a marker for the unambiguous identification of the A tract location. When the A tract and cruciform were placed diametrically opposite, this yielded predominantly nonbranched plectonemic molecules with an extruded cruciform and A tract localized in the terminal loops. For both the curved A tract and mixed sequence nonbent DNA, their localization to an apex increased the angle of bending compared to that expected for DNA unconstrained in solution. This is consistent with increased helical distortion at an apical bend.

Base Sequence↗

PubMed2004

Influence of global DNA topology on cruciform formation in supercoiled DNA.

DNA supercoiling plays an important role in many genetic processes such as replication, transcription, and recombination. Supercoiling provides energy for helix un-pairing and drives the formation of alternative DNA structural transitions, like cruciforms. Supercoiling also allows distant DNA regions to be brought into close proximity through the formation of inter-wound supercoils. Recently, we showed that the inverted repeat-to-cruciform transition acts as a molecular switch, influencing the global topology of a topological plasmid domain. As alternative DNA structures can affect global topology, a corollary hypothesis might be that the localization of a specific DNA sequence within a topological domain may affect the energetics required for formation of an alternative DNA structure. Here, we test this hypothesis and show that the localization of an inverted repeat to an apical position increases the rate of cruciform formation and reduces the superhelical energy required to drive the transition. For this, we created a series of plasmids containing an inverted repeat and an A-tract bent DNA sequence. The A-tract forms a permanent 180 degrees bend irrespective of DNA topology. The inverted repeat and the bent sequence were placed either at six o'clock or nine o'clock positions with respect to each other. Using 2D agarose gel electrophoresis, we show that the six o'clock construct extrudes the cruciform at a lower superhelical density than a control plasmid without the bend. Atomic force microscopy shows that the nine o'clock construct has the propensity to form branched molecules with the cruciform at the end of one branch. These results demonstrate that the localization of sequences within specific regions of a topological domain can affect the energetics of structural transitions as well as the branching structure of the domain. As structural transitions can be involved in biological processes, localization of alternative conformation-forming sequences to specific locations within a domain provides an additional means for gene regulation.

DNA, Superhelical↗

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

Applications of triple-stranded nucleic acid structures to DNA purification, detection and analysis.

Regions of double-stranded (duplex) DNA with purine bases predominantly in one strand and pyrimidine bases in the other may bind oligonucleotides of an appropriate sequence to form triple-stranded (triplex) structures. Oligonucleotide analogs and mimics, such as peptide nucleic acid, may also form stable complexes with duplex DNA. Triplex formation enables the specific targeting of duplex domains. The principles of triplex structures and recent developments in the gene therapeutic and biotechnological applications are briefly reviewed. Adaptations of triplex methodology to molecular diagnostics (DNA purification, detection and analysis) are reviewed in greater detail.

DNA↗

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

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↗

PubMed2001

Transcriptional repression by binding of poly(ADP-ribose) polymerase to promoter sequences.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD+ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4-5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP(-/-) cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.

5' Flanking Region↗

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↗