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At least 649 records · Page 36Linked to original sources
PubMed1983

Sequence-dependent conformation of an A-DNA double helix. The crystal structure of the octamer d(G-G-T-A-T-A-C-C).

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1.8 and 2.25 A, respectively. The molecules adopt and A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.

Base Composition↗
PubMed2003

ATP-dependent selection between single nucleotide and long patch base excision repair.

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.

Adenosine Triphosphate↗
PubMed1996

Detailed characterization of antisense DNA oligonucleotides.

We have developed methods for verification of the structures of novel, chemically synthesized oligonucleotides having alternating methylphosphonate/phosphodiester internucleotide linkages. Matrix-assisted laser desorption ionization mass spectrometry was used to measure the molecular masses of full-length oligonucleotides, failure synthesis products, and degradation products formed by enzymatic and chemical means. These measurements provide detailed structural information, including molecular mass, length, base composition, complete nucleotide sequence, and confirmation of the sugar moieties and internucleotide linkages.

Animals↗
PubMed1988

Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.

Base Composition↗
PubMedDate not supplied

High-resolution structure of a DNA helix containing mismatched base pairs.

The concept of complementary base pairing, integral to the double-helical structure of DNA, provides an effective and elegant mechanism for the faithful transmission of genetic information. Implicit in this model, however, is the potential for incorporating non-complementary base pairs (mismatches) during replication or subsequently, for example, during genetic recombination. As such errors are usually damaging to the organism, they are generally detected and repaired. Occasionally, however, the propagation of erroneous copies of the genome confers a selective advantage, leading to genetic variation and evolutionary change. An understanding of the nature of base-pair mismatches at a molecular level, and the effect of incorporation of such errors on the secondary structure of DNA is thus of fundamental importance. We now report the first single-crystal X-ray analysis of a DNA fragment, d(GGGGCTCC), which contains two non-complementary G X T base pairs, and discuss the implications of the results for the in vivo recognition of base-pair mismatches.

Base Composition↗
PubMed1993

Cooperative and anticooperative binding to a ribozyme.

The effects of guanosine 5'-monophosphate and 2'-deoxyguanosine 5'-monophosphate on the thermodynamics and kinetics of pyrene-labeled 5' exon mimic (pyCUCU) binding to the catalytic RNA (ribozyme) from Tetrahymena thermophila have been determined by fluorescence titration and kinetics experiments at 15 degrees C. pyCUCU binding to L-21 Sca I-truncated ribozyme is weaker by a factor of 5 in the presence of saturating guanosine 5'-monophosphate, whereas it is 4-fold stronger in the presence of saturating 2'-deoxyguanosine 5'-monophosphate. Results from kinetics experiments suggest that anticooperative effects in the presence of guanosine 5'-monophosphate arise primarily from slower formation of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex formed by pyCUCU and GGAGGG of the ribozyme. Conversely, cooperative effects in the presence of 2'-deoxyguanosine 5'-monophosphate arise primarily from slower disruption of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex. Additional experiments suggest that these cooperative and anticooperative effects are not a function of the pyrene label, are not caused by a salt effect, and are not specific to one renaturation procedure for the ribozyme.

Animals↗
PubMed1994

Binding of echinomycin to d(GCGC)2 and d(CCGG)2: distinct stacking interactions dictate the sequence-dependent formation of Hoogsteen base pairs.

Molecular dynamics simulations have been used to explore the behavior of the complexes of echinomycin with the DNA tetramers d(GCGC)2 and d(CCGG)2 in which the terminal bases have been paired according to either a Hoogsteen or a Watson-Crick hydrogen bonding scheme. The energy of the four resulting complexes has been monitored along the dynamics trajectories and the interaction energy between echinomycin and DNA has been decomposed into contributions arising from the planar aromatic systems and the depsipeptide part of the antibiotic. Our calculations predict a large increase in overall stabilization upon protonation of the terminal cytosines and subsequent Hoogsteen pair formation in the complex of echinomycin with d(GCGC)2 but not with d(CCGG)2, in agreement with the experimental evidence [Gao and Patel, Quart. Rev. Biophys. 22, 93-138 (1989)]. The conformational preferences appear to arise mainly from differential stacking interactions in which the electrostatic component is shown to play a dominant role. Differences in hydrogen bonding patterns are also found among the complexes and these are compared in relation to available crystal structures. The binding of echinomycin to DNA appears as a complex process involving many interrelated variables.

Base Composition↗
PubMed1974

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.

Electrophoresis of ribosomal RNA in polyacrylamide-agarose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel electrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.

Barium↗
PubMed1975

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.

Electrophoresis of ribosomal RNA in polyacrylamide-agrose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel elctrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.

Barium↗
PubMed1995

Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides.

A triplex-forming oligopyrimidine has been attached at its 5'-end to a photoreactive psoralen derivative and used to target a sequence which forms part of the coding region of the human aromatase gene. The 20 base pair sequence is not a perfect triplex target since it contains three pyrimidine interruptions within the purine-rich strand. Despite this, we have detected triplex-directed photoadduct formation at pH 7.0 between the psoralen-linked oligonucleotide and a 30mer duplex representing the aromatase target. Photoadduct formation was found to be sensitive to pH, temperature, cation concentration and the base composition of the third strand. By varying the base sequence of the target duplex around the psoralen intercalation site, we have characterised the site and mode of psoralen intercalation. The attached psoralen has been found to intercalate at the triplex-duplex junction with a strong preference for one orientation. We have shown that the psoralen will bind at the junction even when there is a preferred TpA step at an adjacent site. We have also compared the binding affinity and photoreactivity of oligodeoxyribonucleotides linked to two different psoralen derivatives and found differences in the rate of crosslinking and the extent of crosslink formation. Finally, we have examined oligodeoxyribonucleotides which are attached to psoralen by polymethylene linkers of different lengths.

Aromatase↗
PubMed1995

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing the lac repressor-operator complex.

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCl, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 25 degrees C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCl to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Kobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution on the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system.

Base Composition↗
PubMed1996

Quinazoline-2,4(1H,3H)-dione as a substitute for thymine in triple-helix forming oligonucleotides: a reassessment.

A major limitation in triple-helix formation arises from the weak energy of interaction between the third strand and the double-stranded target. We tried to increase the stacking interaction contribution within the third strand by extending the aromatic domain of thymine. We report here the use of 2,4-quinazolinedione as a substitute for thymine in the canonical TA*T triplet. The synthesis and the characterization of the quinazoline beta nucleoside Q and of its phosphoramidite derivative is described. Triple-helix- forming oligonucleotides incorporating Q have been prepared and their ability to form triplexes has been evaluated by UV-monitored thermal denaturation measurements. The introduction of one or multiple Q residues, either contiguous or remote from each other, slightly destabilized triple-stranded structures, whatever the nucleic acid base composition (pyrimidine or GT) of the third strand.

Drug Stability↗
PubMed2002

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Base Pair Mismatch↗
PubMed1979

The use of rifampicin to evaluate tRNA transcriptional organization in Escherichia coli.

The antibiotic rifampicin, which in prokaryotes inhibits the initiation of RNA synthesis but not the completion of nascent strands, was used to explore tRNA gene transcriptional organization in Escherichia coli. Cultures were grown in [32P] orthophosphate to constant specific radioactivity and labeled with [3H] uridine in the presence of rifampicin. Numerous tRNA species then were isolated by polyacrylamide gel electrophoresis and their 3H/32P ratios determined; these ratios, following correction for the base compositions of the tRNAs, should reflect the distances of the corresponding tRNA genes from their promoters. Individual tRNA species were identified, where possible, by oligonucleotide fingerprint analysis. Observed isotopic ratios were correlated with promoter-gene distances, measured in nucleotides, using the nucleotide sequence of the 16S ribosomal RNA gene as a reference. The protocols developed should be applicable to most prokaryotes.

Base Sequence↗
PubMed1980

Loopstructures in synthetic oligodeoxynucleotides.

A comparative nuclear magnetic resonance study of the hydrogen-bonded imino protons in a series of synthetic DNA fragments is presented. The fragments ATCCTA(Tn)TAGGAT are in principle capable of forming either a self-complementary hairpin loop structure (monomer form) or an interior loop structure (dimeric form). It has been shown, that for n = 1 only the dimer structure is present in aqueous solution, whereas the exclusive existence of the hairpin loop structure is indicated for n = 3, 4 & 5. Surprisingly, for n = 2 two different structures appear to be present in solution. Concentration studies show that both monomers and dimers exist side by side in this case. Hairpins as well as interior loops form extra "melting sites" in addition to the wellknown fraying phenomenon at the terminus of the double helix.

Base Composition↗
PubMed2002

Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection.

Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have exploited these properties of LNA probes to develop a new method for single nucleotide polymorphism genotyping. In this method, very short (hexamer or heptamer) LNA probes are labeled with either rhodamine or hexachlorofluorescein (HEX), and their hybridization to target DNAs is followed by measuring the fluorescence polarization (FP) of the dyes. The formation of perfectly complementary double-stranded hybrids gives rise to significant FP increases, whereas the presence of single mismatches results in very small or no changes of this parameter. Multiplexing of the assay can be achieved by using differentially labeled wild-type and mutant specific probes in the same solution. The method is homogeneous, and because of the use of extremely short LNA probes, the generation of a universal set of genotyping reagents is possible.

Base Composition↗
PubMed2003

Improved net protein balance, lean mass, and gene expression changes with oxandrolone treatment in the severely burned.

OBJECTIVE: To determine the effects of the anabolic agent oxandrolone on muscle protein and gene expression in severely burned children. SUMMARY BACKGROUND DATA: The authors previously showed that oxandrolone increased net muscle protein synthesis in emaciated burned patients receiving delayed treatment for severe burns. They hypothesized that similar effects would be seen in those treated early after burn. METHODS: Thirty-two severely burned children were enrolled in a prospective randomized trial. Subjects underwent studies to assess leg protein net balance 5 days after the first excision and grafting procedure. Immediately after these studies, treatment with placebo (n = 18) or 0.1 mg/kg oxandrolone (n = 14) twice a day was started. One week after this, another net balance study was performed in each subject. Body weights and total body potassium counting were used to determine body compositional changes. Muscle biopsies were taken 1 week after treatment in oxandrolone subjects to examine gene expression changes with gene array (12,600 genes). RESULTS: Protein net balance did not change in the placebo group, while oxandrolone-treated subjects had a significant improvement. Body weights and fat free mass significantly decreased in the placebo group, while no changes were found in the oxandrolone-treated subjects. Expression changes were seen in 14 genes in the oxandrolone group compared to placebo. Some of these included myosin light chain (+2.7-fold change), tubulin (+2.3), calmodulin (-2.3), and protein phosphatase I inhibitor (-2.8). CONCLUSIONS: Oxandrolone improves protein net balance and lean mass in the severely burned. These changes are associated with increased gene expression for functional muscle proteins.

Amino Acids↗
PubMed1990

Site-specific transposition of insertion sequence IS630.

IS630 is a 1.15-kilobase sequence in Shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. To assess its transposition, we constructed composite elements containing inverted copies of IS630 flanking a drug resistance gene. We found that these composite elements transposed to plasmid ColE1 in Escherichia coli. DNA sequencing showed that transposition was, in all cases, to the dinucleotide sequence 5'-TA-3'. There were two preferred insertion sites which corresponded to the TA sequences in the inverted repeats of a 13-base-pair stem region of the [rho]-dependent transcription terminator. IS630 is flanked by TA, and nucleotide substitution by in vitro mutagenesis at these ends did not affect transposition activity of a composite element or its ability to insert preferentially into TA within the 13-base-pair inverted repeat sequences or to duplicate the target sequence.

Base Composition↗