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At least 199 records · Page 11Linked to original sources
PubMed2002

Triplex and quadruplex DNA structures studied by electrospray mass spectrometry.

DNA triplex and quadruplex structures have been successfully detected by electrospray ionization mass spectrometry (ESI-MS). Circular dichroism and UV-melting experiments show that these structures are stable in 150 mM ammonium acetate at pH 7 for the quadruplexes and pH 5.5 for the triplexes. The studied quadruplexes were the tetramer [d(TGGGGT)](4), the dimer [d(GGGGTTTTGGGG)](2), and the intramolecular folded strand dGGG(TTAGGG)(3), which is an analog of the human telomeric sequence. The absence of sodium contamination allowed demonstration of the specific inclusion of n - 1 ammonium cations in the quadruplex structures, where n is the number of consecutive G-tetrads. We also detected the complexes between the quadruplexes and the quadruplex-specific drug mesoporphyrin IX. MS/MS spectra of [d(TGGGGT)](4) and the complex with the drug are also reported. As the drug does not displace the ammonium cations, one can conclude that the drug binds at the exterior of the tetrads, and not between them. For the triplex structure the ESI-MS spectra show the detection of the specific triplex, at m/z values typically higher than those typically observed for duplex species. Upon MS/MS the antigene strand, which is bound into the major groove of the duplex, separates from the triplex. This is the same dissociation pathway as in solution. To our knowledge this is the first report of a triplex DNA structure by electrospray mass spectrometry.

Acetates↗
PubMed2000

Poly(dA.dT) sequences exist as rigid DNA structures in nucleosome-free yeast promoters in vivo.

Poly(dA.dT) sequences (T-tracts) are abundant genomic DNA elements with unusual properties in vitro and an established role in transcriptional regulation of yeast genes. In vitro T-tracts are rigid, contribute to DNA bending, affect assembly in nucleosomes and generate a characteristic pattern of CPDs (cyclobutane pyrimidine dimers) upon irradiation with UV light (UV photofootprint). In eukaryotic cells, where DNA is packaged in chromatin, the DNA structure of T-tracts is unknown. Here we have used in vivo UV photofootprinting and DNA repair by photolyase to investigate the structure and accessibility of T-tracts in yeast promoters (HIS3, URA3 and ILV1). The same characteristic photofootprints were obtained in yeast and in naked DNA, demonstrating that the unusual T-tract structure exists in living cells. Rapid repair of CPDs in the T-tracts demonstrates that these T-tracts were not folded in nucleosomes. Moreover, neither datin, a T-tract binding protein, nor Gcn5p, a histone acetyltransferase involved in nucleosome remodelling, showed an influence on the structure and accessibility of T-tracts. The data support a contribution of this unusual DNA structure to transcriptional regulation.

Acetyltransferases↗
PubMed2004

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex.

The causes of spontaneous chromosomal translocations in somatic cells of biological organisms are largely unknown, although double-strand DNA breaks are required in all proposed mechanisms. The most common chromosomal abnormality in human cancer is the reciprocal translocation between chromosomes 14 and 18 (t(14;18)), which occurs in follicular lymphomas. The break at the immunoglobulin heavy-chain locus on chromosome 14 is an interruption of the normal V(D)J recombination process. But the breakage on chromosome 18, at the Bcl-2 gene, occurs within a confined 150-base-pair region (the major breakpoint region or Mbr) for reasons that have remained enigmatic. We have reproduced key features of the translocation process on an episome that propagates in human cells. The RAG complex--which is the normal enzyme for DNA cleavage at V, D or J segments--nicks the Bcl-2 Mbr in vitro and in vivo in a manner that reflects the pattern of the chromosomal translocations; however, the Mbr is not a V(D)J recombination signal. Rather the Bcl-2 Mbr assumes a non-B-form DNA structure within the chromosomes of human cells at 20-30% of alleles. Purified DNA assuming this structure contains stable regions of single-strandedness, which correspond well to the translocation regions in patients. Hence, a stable non-B-DNA structure in the human genome appears to be the basis for the fragility of the Bcl-2 Mbr, and the RAG complex is able to cleave this structure.

Animals↗
PubMed1992

The human insulin gene linked polymorphic region exhibits an altered DNA structure.

Regulation of transcription of the human insulin gene appears to involve a series of DNA sequences in the 5' region. Hypersensitivity to DNA structural probes has previously been demonstrated in regulatory regions of cloned genomic DNA fragments, and been correlated with gene activity. To investigate the structure of the DNA in the human insulin gene, bromoacetaldehyde and S1 nuclease were reacted with a supercoiled plasmid containing a 5kb genomic insulin fragment. Both probes revealed the human insulin gene linked polymorphic region (ILPR), a region (-363) upstream of the transcriptional start site which contains multiple repeats of a 14-15mer oligonucleotide with the consensus sequence ACAGGGGT(G/C)(T/C)GGGG, as the major hypersensitive site. Fine mapping and electron microscopic analysis both show a very different behaviour of the two DNA strands in the region of the ILPR and suggest the G-rich strand may be adopting a highly structured conformation with the complementary strand remaining largely single stranded.

Base Sequence↗
PubMed1990

Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure. II. NaCl-dependence of DNA flexibility and helical repeat.

In this work, we demonstrate that it is possible to determine the molar cyclization factor jM from single ligation reactions in which both circular and linear dimer DNA species are formed concurrently from linear monomers. This approach represents a significant improvement over previous methods, in which jM is evaluated from the ratio of the rate constants for two separate processes; namely (1) the cyclization of linear DNA and (2) the association of two linear molecules to form linear dimers. Determination of jM for a 366 base-pair molecule yields 5.8 X 10(-8) M, in close agreement with the value of 5.6 X 10(-8) M determined by Shore et al. for the same molecule. Using the current approach for the determination of jM, we have investigated the dependence on NaCl concentration (0 to 162 mM-NaCl, 1 mM-MgCl2) of both the lateral and torsional flexibilities of DNA. The principal observation is that both quantities are essentially constant over the above range of NaCl concentrations, with the persistence length P approximately 450 (+/- 15) A, and the torsional elastic constant C approximately 2.0 (+/- 0.2) X 10(-19) erg cm. These observations are in accord with the previous theoretical prediction that P becomes essentially independent of NaCl concentration above 10 to 20 mM. We have examined the dependence of the helical repeat of DNA on NaCl concentration over the above range, and have found the value of 10.44 base-pairs per turn to be essentially constant over that range. This last result suggests that earlier studies have overestimated the dependence of DNA helical twist on salt concentration.

Base Sequence↗
PubMed2006

Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3' terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes.

Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3' phosphodiesterase and 3'-5' exonuclease activities. The 3'-5' exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase beta. Here, we examine the removal of matched and mismatched dNMP from the 3' terminus of the 3'-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3' terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3' terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3'-5' exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5' margin of the nick. The 3'-5' exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5' margin of the nick as well as for flapped and recessed DNAs.

DNA-(Apurinic or Apyrimidinic Site) Lyase↗
PubMed1996

Computation of DNA structural variability--a new predictor of DNA regulatory regions.

MOTIVATION: Local separation of the two strands of the DNA duplex is an essential step in important biological activities, including the initiation of transcription and replication. This conformational transition occurs in response to imposed stresses, which are stringently regulated in vivo. RESULTS: This paper describes two computational methods to analyse this phenomenon: an approximate statistical mechanical method and a Monte Carlo sampling technique. Analysis of genomic DNA sequences shows that sites of predicted duplex destabilization are closely associated with regions regulating transcription, with the most destabilized sites coinciding with 3' gene termini. Experimental results supporting this conclusion are described. The incorporation of this technique into computational searches for regulatory regions is discussed. AVAILABILITY: Because the programs implementing these calculations are complex to use and not completely developed, they have not been released yet. However, the author will analyze any DNA sequences upon request. Sequences may be submitted electronically to benham/msvax.mssm.edu.

DNA↗
PubMed2004

Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures.

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.

Adenosine Triphosphatases↗
PubMed1999

DNA structural transitions within the PKD1 gene.

Autosomal dominant polycystic kidney disease (ADPKD) affects over 500 000 Americans. Eighty-five percent of these patients have mutations in the PKD1 gene. The focal nature of cyst formation has recently been attributed to innate instability in the PKD1 gene. Intron 21 of this gene contains the largest polypurine. polypyrimidine tract (2.5 kb) identified to date in the human genome. Polypurine.polypyrimidine mirror repeats form intramolecular triplexes, which may predispose the gene to mutagenesis. A recombinant plasmid containing the entire PKD1 intron 21 was analyzed by two-dimensional gel electrophoresis and it exhibited sharp structural transitions under conditions of negative supercoiling and acidic pH. The superhelical density at which the transition occurred was linearly related to pH, consistent with formation of protonated DNA structures. P1 nuclease mapping studies of a plasmid containing the entire intron 21 identified four single-stranded regions where structural transitions occurred at low superhelical densities. Two-dimensional gel electrophoresis and chemical modification studies of the plasmid containing a 46 bp mirror repeat from one of the four regions demonstrated the formation of an H-y3 triplex structure. In summary, these experiments demonstrate that a 2500 bp polypurine.polypyrimidine tract within the PKD1 gene is capable of forming multiple non-B-DNA structures.

Base Sequence↗
PubMedDate not supplied

Biological fingerprinting analysis by liquid chromatography/mass spectrometry for evaluation of DNA structural selectivity of multiple compounds in natural products.

A method by combination of centrifugal ultrafiltration (CUF) sampling with liquid chromatography-mass spectrometry (LC-MS) analysis was established to evaluate the DNA structure and sequence selectivity of the multiple compounds in a small molecule library. The developed method was applied to analyze the extracts of natural products Coptis chinensis Franch and Rheum palmatum (L.). From the obtained biological fingerprinting chromatograms, 7 compounds in C. chinensis Franch and 11 in R. palmatum (L.) were screened out as DNA binding agents. Most of these compounds were identified by standards and LC-MS analysis after the sample pretreatment with the DNA immobilized cartridge. DNA structural binding preference of the multiple active compounds in these two extracts was then evaluated simultaneously without purification.

Binding Sites↗
PubMedDate not supplied

[Viscosimetric analysis of the dynamics of alkaline lysis of mammalian cell nuclei as a method of studying radiation damage to DNA structures].

The method is proposed for the analysis of DNA viscosity during alkaline lysis of rat thymocyte nuclei which permits to reveal early changes in the DNA structure at doses of 0.5 to 1 Gy. Doses from 0.5 to 30 Gy were chosen for the experiment. The changes in the lysis dynamics, the decrease in the intrinsic viscosity maximum [eta], and the increase in the alkaline lysis rate were shown to be a function of radiation dose.

Animals↗
PubMed1999

Mapping of eukaryotic DNA topoisomerase I catalyzed cleavage without concomitant religation in the vicinity of DNA structural anomalies.

Sensitive sites for covalent trapping of eukaryotic topoisomerase I at DNA structural anomalies were mapped by a new method using purified enzyme and defined DNA substrates. To insure that the obtained topoisomerase I trapping patterns were not influenced by DNA sequence variations, a single DNA imperfection was placed centrally within a homonucleotide track. Mapping of topoisomerase I-mediated irreversible cleavage sites on homopolymeric DNA substrates containing mismatches showed trapping of the enzyme in several positions in close vicinity of the DNA imperfection, with a strong preference for the 5' junction between the duplex DNA and the base-pairing anomaly. On homopolymeric DNA substrates containing a nick, sites of topoisomerase I-mediated cleavage on the intact strand were located just opposite to the nick and from one to ten nucleotides 5' to the nick. Sites of enzyme-mediated cleavage next to a nick and an immobile single-stranded branch were located 5' to the strand interruption in distances of two to six nucleotides and two to ten nucleotides, respectively. Taken together these findings suggest that covalent trapping of topoisomerase I proceeds at positions adjacent to mismatches, nicks and single-stranded branches, where the cleavage reaction is allowed and the ensuing ligation reaction prevented. In principle, the developed interference method might be of general utility to define topoisomerase-DNA interactions relative to different types of structural anomalies.

Autoradiography↗
PubMed2000

A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints.

To further dissect the genetic differences between the checkpoint pathway following S-phase cdc arrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase delta mutant, cdc20-M10. One such checkpoint mutant, rad17-d14, was found to display the cut phenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1 mutant. Unlike chk1 , rad17-d14 was not sensitive to UV irradiation. Interestingly, the ionising radiation sensitivity of rad17-d14 was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G(2) phase. Biochemical analysis attributes the checkpoint defects of rad17-d14 to the failure to phosphorylate the checkpoint effector Chk1p. To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed. Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase. This interaction occurred irrespective of the arrest with cdc20-M10 and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.

Alleles↗
PubMed2001

HMG box A in HMG2 protein functions as a mediator of DNA structural alteration together with box B.

Nonhistone protein HMG2, like HMG1, binds with B-DNA in a sequence-nonspecific manner and causes structural alterations in DNA such as bending, kinking and unwinding. Here, we studied the functions of HMG2 domains in the DNA structural alteration and modulation by using various HMG2 peptides, and we demonstrated several new findings. The HMG box itself as a DNA-binding motif may have the basic function of inducing curvature, resulting in the apparent DNA bending in the DNA cyclization assay, but not of abruptly kinking DNA. The DNA-binding activity of HMG box B, which is enhanced by the presence of box A, together with the flanking regions of box B, causes DNA bending accompanying the kinking of the DNA main chain. The DNA unwinding accompanied by DNA kinking diminishes cruciform structures in supercoiled DNA. Analysis using mutant peptides for box A confirmed that box A in HMG2 functions as a mediator of DNA structural alteration together with box B. The present studies on the functional properties of the respective regions of HMG2 may help to elucidate the protein function.

Amino Acid Motifs↗
PubMed1997

High order DNA structure as inferred by optical fluorimetry and scanning calorimetry.

New quantitative insights on the native high order chromatin-DNA structure existing within interphase nuclei are obtained by monitoring the effects of two common well-characterized fixatives, glutaraldehyde and ethanol/acetic acid mixture, at the level of the intranuclear DNA distribution and structures. Reproducible distinct levels of DNA fluorescence intensity and their intranuclear distribution are apparent in unfixed and fixed thymocytes by using DAPI and quantitative optical microscopy based on a charge coupled device. The fluorescent histograms correlated with the calorimetric thermograms on the very same thymocytes fixed and unfixed, establish an unequivocal baseline for the different levels of structural organization of the chromatin within the intact nucleus; namely their number, DNA packing ratio and fiber diameter. A systematic comparison among all the numerous models, being so far proposed for the quinternary and quaternary levels of DNA folding, to identifies the rope or ribbon-like and the chromonema as the ones that best fit with the in situ distribution.

Animals↗
PubMed1996

A triplex DNA structure of the polypyrimidine: polypurine stretch in the 5' flanking region of the sea urchin arylsulfatase gene.

Previously we reported that a long (522 bp) polypyrimidine: polypurine stretch in the 5' flanking region of the arylsulfatase gene of the sea urchin, Hemicentrotus pulcherrimus, took an unusual, perhaps triplex, DNA structure, when subjected to an acidic pH (pH 5) (Yamamoto et al., 1994). In the present study we have isolated a polypyrimidine: polypurine containing fragment from the arylsulfatase gene and surveyed the sensitivities of the polypyrimidine: polypurine stretch to base modification by diethylpyrocarbonate and osmium tetroxide under various levels of negative supercoiling. Based on the sensitivity of highly negatively supercoiled DNA to these base-modifying reagents, we conclude that, when highly negatively supercoiled, the polypyrimidine: polypurine stretch can take a triplex DNA structure even at a neutral pH and under physiological ionic strength in the presence of Mg2+.

Amino Acid Sequence↗