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V N Potaman

Publications and source records attributed to V N Potaman.

At least 19 recordsLinked to original sources
PubMed2004

Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH.

More than 15 human genetic diseases have been associated with the expansion of trinucleotide DNA repeats, which may involve the formation of non-duplex DNA structures. The slipped-strand nucleation of duplex DNA within GC-rich trinucleotide repeats may result in the changes of repeat length; however, such a mechanism seems less likely for the AT-rich (GAA)n*(TTC)n repeats. Using two-dimensional agarose gels, chemical probing and atomic force microscopy, we characterized the formation of non-B-DNA structures in the Friedreich ataxia-associated (GAA)n*(TTC)n repeats from the FRDA gene that were cloned with flanking genomic sequences into plasmids. For the normal genomic repeat length (n = 9) our data are consistent with the formation of a very stable protonated intramolecular triplex (H-DNA). Its stability at pH 7.4 is likely due to the high proportion of the T.A.T triads which form within the repeats as well as in the immediately adjacent AT-rich sequences with a homopurine. homopyrimidine bias. At the long normal repeat length (n = 23), a family of H-DNAs of slightly different sizes has been detected. At the premutation repeat length (n = 42) and higher negative supercoiling, the formation of a single H-DNA structure becomes less favorable and the data are consistent with the formation of a bi-triplex structure.

AT Rich Sequence↗
PubMed2004

DNA-binding properties of poly(ADP-ribose) polymerase: a target for anticancer therapy.

Poly(ADP-ribose) polymerization is a unique post-translation protein modification that utilizes an ADP-ribose moiety from NAD+ to form long and branched polymers attached via glutamic acid residues to nuclear acceptor proteins. The corresponding enzyme, poly(ADP-ribose) polymerase (PARP-1), is a zinc finger-containing protein, which allows PARP-1 binding to either double- or single-strand DNA breaks. The catalytic activity of PARP-1 is strictly dependent on the presence of strand breaks in DNA, and is modulated by the level of automodification. PARP-1 is regarded as an intracellular sensor for DNA strand breaks, and its function has been implicated in cellular processes that require DNA cleavage and rejoining reactions, such as DNA replication, recombination and repair. Recent studies have also implicated PARP-1 in the regulation of gene expression through modification of transcription factors by poly(ADP-ribosyl)ation or its direct binding to gene-regulating DNA sequences. The latter is attributable to PARP's ability to recognize and bind to various structural discontinuities in the DNA duplex in the absence of DNA strand breaks, such as three- or four-way junctions, bent DNA, and base unpaired regions. Cumulatively, these findings indicate that PARP-1 plays a pivotal role in the maintenance of the genome integrity during the normal functioning of eukaryotic cells as well as in the cellular responses to DNA damage, and that PARP-DNA interactions are indispensable for PARP function. This review summarizes the data on DNA-binding properties of PARP-1 and relates them to the development of strategies for sensitizing tumor cells to genotoxic treatments.

Animals↗
PubMed2001

The structure of intramolecular triplex DNA: atomic force microscopy study.

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.

Base Sequence↗
PubMed2001

Involvement of the nucleotide excision repair protein UvrA in instability of CAG*CTG repeat sequences in Escherichia coli.

Several human genetic diseases have been associated with the genetic instability, specifically expansion, of trinucleotide repeat sequences such as (CTG)(n).(CAG)(n). Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single strands. Subsequently, these loops or hairpins may be recognized and processed by DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repair-deficient Escherichia coli strains (using a genetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. Moreover, loops containing 23 CTG repeats were less efficiently excised from heteroduplex plasmids after their transformation into the uvrA(-) strain. As a result, an increased proportion of plasmids containing the full-length repeat were recovered after the replication of heteroduplex plasmids containing unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a K(d) of about 10-20 nm, which is an affinity about 2 orders of magnitude higher than that of UvrA bound to the control substrates containing (CTG)(n).(CAG)(n) in the linear form. These results suggest that UvrA is involved in triplet repeat instability in cells. Specifically, UvrA may bind to loops formed during replication slippage or in slipped strand DNA and initiate DNA repair events that result in repeat deletion. These results imply a more comprehensive role for UvrA, in addition to the recognition of DNA damage, in maintaining the integrity of the genome.

Adenosine Triphosphatases↗
PubMed2000

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies.

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.

DNA↗
PubMed2000

A cruciform structural transition provides a molecular switch for chromosome structure and dynamics.

The interaction between specific sites along a DNA molecule is often crucial for the regulation of genetic processes. However, mechanisms regulating the interaction of specific sites are unknown. We have used atomic force microscopy to demonstrate that the structural transition between cruciform conformations can act as a molecular switch to facilitate or prevent communication between distant regions in DNA. Cruciform structures exist in vivo and they are critically involved in the initiation of replication and the regulation of gene expression in different organisms. Therefore, structural transitions of the cruciform may play a key role in these processes.

Base Pairing↗
PubMed1999

Structure of branched DNA molecules: gel retardation and atomic force microscopy studies.

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.

Base Sequence↗
PubMed1999

Overcoming a barrier for DNA polymerization in triplex-forming sequences.

Folded structures in the DNA template, such as hairpins and multi-stranded structures, often serve as pause and arrest sites for DNA polymerases. DNA polymerization is particularly difficult on mirror-repeated homopurine.homopyrimidine templates where triple-stranded (triplex) structures may form between the nascent and folded template strands. In order to use a linear PCR amplification approach for the structural analysis of DNA in mirror-repeated sequences we modified a conventional protocol. The barrier for DNA synthesis can be eliminated using an oligonucleotide that hybridizes with the template to prevent its folding and is subsequently displaced by the progressing polymerase. The described approach is potentially useful for sequencing and analysis of chemical adducts and point mutations in a variety of sequences prone to the formation of folded structures, such as long hairpins and quadruplexes.

Animals↗
PubMed1998

Stabilization of intramolecular triple/single-strand structure by cationic peptides.

For better comprehension of possible physiological roles of triple-helical DNA structures, it is important to understand if the proteins can stabilize intramolecular triplex (H-DNA). One plausible mode of stabilization is through the neutralization of electrostatic repulsion of negatively charged phosphates in the three DNA strands by positively charged arginine and lysine residues of a bound protein. To gain an insight into interactions between H-DNA and cationic protein domains, we examined the effect of Lys- and Arg-rich oligopeptides on the B-DNA to H-DNA transition. These oligopeptides as well as another type of polycation, spermine, shifted the equilibrium toward H-DNA. These polycations introduced little change in DNA superhelicity, so that an increase in torsional stress was not responsible for facilitated H-DNA formation. Competing influences of polycations and monovalent cations suggest a significant involvement of electrostatic interactions in H-DNA stabilization. The Arg-rich peptides are more effective in H-DNA stabilization than the Lys-rich ones. However, as inferred from experiments on intermolecular complexes, this is not due to a better stabilization of triple helix or destabilization of double helix. It is possible that Arg-rich peptides interact with the unpaired single strand in H-DNA and stabilize its unpaired conformation.

Arginine↗
PubMed1998

Structure and dynamics of supercoil-stabilized DNA cruciforms.

Understanding DNA function requires knowledge of the structure of local, sequence-dependent conformations that can be dramatically different from the B-form helix. One alternative DNA conformation is the cruciform, which has been shown to have a critical role in the initiation of DNA replication and the regulation of transcription in certain systems. In addition, cruciforms provide a model system for structural studies of Holliday junctions, intermediates in homologous DNA recombination. Cruciforms are not thermodynamically stable in linear DNA due to branch point migration, which makes their study using many biophysical techniques problematic. Atomic Force Microscopy (AFM) was applied to visualize cruciforms in negatively supercoiled plasmid DNA. Cruciforms are seen as clear-cut extrusions on the DNA filament with the lengths of the arms consistent with the size of the hairpins expected from a 106 bp inverted repeat. The cruciform exists in two different conformations, an extended one with the angle of ca. 180 degrees between the hairpin arms and a compact, X-type conformation, with acute angles between the hairpin arms and the main DNA strands. The ratio of molecules with the different conformations of cruciforms depends on ionic conditions. In the presence of high salt or Mg cations, a compact, X-type conformation is highly preferable. Remarkably, the X-conformation was highly mobile allowing the cruciform arms to adopt a parallel orientation. The structure observed is consistent with a model of the Holliday junction with a parallel orientation of the exchanging strands.

DNA, Superhelical↗
PubMed1996

Formation of a combined H-DNA/open TATA box structure in the promoter sequence of the human Na,K-ATPase alpha2 gene.

Structural variation of DNA within the promoter of the human Na, K-ATPase alpha2 gene, which contains a 35-base pair (bp) homopyrimidine.homopurine (Py.Pu) tract adjacent to a TATA box has been studied. The Py.Pu tract contains a 26-bp quasi-mirror repeat sequence with a potential for intramolecular triplex formation. As analyzed by two-dimensional agarose gel electrophoresis, a plasmid containing 151 bp of the promoter sequence including the 35-bp Py.Pu tract undergoes structural transitions under moderately acidic pH. Chemical probing with chloroacetaldehyde, dimethyl sulfate, and potassium permanganate is consistent with the formation of triplex DNA within the Py.Pu tract at native superhelical density as isolated from Escherichia coli. Chemical probing was used to determine a supercoil dependence for the formation of this combined unwound structure. At the superhelical density sufficient to locally unwind DNA, an H-y3 isomer of intermolecular triplex likely forms. However, at higher superhelical tension an H-y5 structure forms in the Py.Pu tract, and with increasing supercoiling the local DNA unwinding extends into the abutting TATA box. The H-y5/open TATA box combination structure might be favorable at higher superhelical densities since it relaxes more supercoils. The possible involvement of the H-y5/open TATA box structure in transcription is discussed.

Base Sequence↗
PubMed1995

Stabilization of triple-helical nucleic acids by basic oligopeptides.

Intermolecular triplex DNA is stabilized by metal cations and polyamines which reduce repulsion between the negatively charged phosphates of the three nucleic acid strands. We use a quantitative chemical-probing assay involving protection of duplex guanines in a homopyrimidine.homopurine (Py.Pu) sequence from dimethyl sulfate modification to study effects of basic oligopeptides on the stability of triplex DNA. An intermolecular protonated pyrimidine.purine.pyrimidine (Py.Pu*Py) triplex formed readily between a duplex DNA region and a 14-mer pyrimidine triplex-forming oligonucleotide (TFO) at pH 5. The triplex was stabilized at pH by the addition of magnesium ions. In the presence of spermine and lysine-rich peptides, the intermolecular triplex was stabilized up to pH 6.5-7.0. The effective peptide concentration required for stabilization was 10(-5)-10(-2) M. Of the basic peptides studied, pentalysine (Lys-Lys-Lys-Lys-Lys) was the most effective triplex stabilizer. It was effective at concentrations which are lower than those required for Lys-Gly-Lys-Gly-Lys and Lys-Ala-Lys-Ala-Lys and are similar to active concentrations of spermine. Basic peptides were more effective at stabilizing a Py.Pu*Py triplex than a pyrimidine.purine.purine (Py.Pu*Pu) triplex. At 1 mM, Lys-Lys-Lys-Lys-Lys stabilized the Py.Pu*Pu triplex at a level comparable to stabilization by Mn2+ and spermine, whereas Lys-Gly-Lys-Gly-Lys and Lys-Ala-Lys-Ala-Lys resulted in weaker TFO binding. The concentration of TFOs required to form triplex DNA were significantly reduced in the presence of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed1995

Oligonucleotide model with non-identical complementary strands for chromatographic studies of structure-dependent photosusceptibility.

In a previous work, we used a quantitative chromatographic analysis of two self-complementary oligonucleotides to correlate the conformational differences between the oligonucleotide duplexes and photochemical susceptibilities of constituent oligomers. In this work we describe a new double-stranded oligonucleotide model with non-identical complementary strands. To separately analyze photoproducts in two strands, one of them is used in a partially protected form (the hydrophobic 5'-dimethoxytrityl group uncleaved). Using a reversed-phase column, the oligomers and products of their UV photomodification are separated into two groups of peaks. This facilitates the quantitation of photoproducts in each of the complementary strands. Three 15-mer oligonucleotides, 5'-TTTTTAT-TAAATATA-3' (F5), 5'-AAAAATAATTTATAT-3' (F6) and 5'-TATATTTAATAAAAA-3' (F7) form the parallel-stranded (ps) F5.F6 and the ordinary antiparallel-stranded (aps) F5.F7 duplexes. For these particular sequences, the rate of cyclobutane thymine dimer formation in the ps DNA has been estimated as ca. 1.5-2 times that in the ordinary aps DNA.

Base Sequence↗
PubMed1995

Radiation and thermal stabilities of adenine nucleotides.

We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

Adenine Nucleotides↗
PubMed1994

Stability of peptide nucleic acids in human serum and cellular extracts.

The stability of a new type of DNA mimic, peptide nucleic acid (PNA) in human blood serum, Eschericia coli and Micrococcus luteus extracts and nuclear and cytoplasmic extracts from mouse Ehrlich ascites tumor cells was investigated using HPLC analysis. Under conditions that caused complete cleavage of a control peptide, adrenocorticotropic hormone fragment 4-10, no significant degradation of the PNAs, H-T10-LysNH2 and H-TGTACGTCACAACTA-NH2, could be detected. Similarly, PNA H-T5-LysNH2 was found to resist attack by fungal proteinase K or porcine intestinal mucosa peptidase at concentrations exceeding those necessary to completely degrade a control peptide, H-Phe-Trp-Tyr-Cys-Phe-Trp-Tyr-Lys-Phe-Trp-Tyr-Lys-OH, by at least 1000- and 30-fold, respectively. Thus PNA is expected to have sufficient biostability to be used as a drug.

Amino Acid Sequence↗
PubMed1994

Divalent metal cations upon coordination to the N7 of purines differentially stabilize the PyPuPu DNA triplex due to unequal Hoogsteen-type hydrogen bond enhancement.

The PyPuPu triplexes consisting of CG*G triads are stabilized by alkaline earth cations (Ca2+, Mg2+) and transition metal cations (Mn2+, Co2+, Ni2+, Zn2+, Cd2+), while similar triplexes including TA*A triads are stabilized only by transition metal cations. We hypothesize that such a differential triplex stabilization by divalent metal cations can be the consequence of their coordination to the N7 of the third strand purines with concomitant polarization effects on the bases resulting in unequal Hoogsteen-type hydrogen bond enhancement.

Cations, Divalent↗