Biomedical subjects

L J Maher

Publications and source records attributed to L J Maher.

At least 37 records · Page 2Linked to original sources

PubMed1998

Effects of phosphate neutralization on the shape of the AP-1 transcription factor binding site in duplex DNA.

Previous electrophoretic experiments suggest that the AP-1 site in duplex DNA bends in response to the pattern of amino acid charges distal to the basic region in bound bZIP proteins. The extent and direction of apparent DNA bending are consistent with the prediction that DNA will collapse locally upon asymmetric phosphate charge neutralization. To prove that asymmetric phosphate neutralization could produce the observed degree of DNA bending, the present experiments partially substitute anionic phosphate diesters in the AP-1 site with various numbers of neutral methylphosphonate linkages. DNA bending is induced toward the neutralized face of DNA. The degree of DNA bending induced by methylphosphonate substitution (approximately 3.5 degrees per neutralized phosphate) is comparable to that induced by GCN4 variants carrying increasing numbers of additional basic amino acids. It is plausible, therefore, that asymmetric phosphate neutralization is the cause of DNA bending in such complexes.

Binding Sites↗

PubMed1998

Characterization of a polypurine/polypyrimidine sequence upstream of the mouse metallothionein-I gene.

A 128 base pair long homopurine/homopyrimidine (R/Y) element is located approximately 1.2 kb upstream of the transcription start point of the mouse metallothionein-I ( MT-I ) gene. We present a detailed in vitro structural characterization of the MT-I R/Y sequence as determined by enzymatic and chemical probes. An approximately 190 bp fragment containing the MT-I R/Y sequence was subcloned into a recombinant vector. Low resolution analysis with S1 nuclease indicates that DNA in this region was unpaired in supercoiled plasmids treated at low pH. High resolution mapping with chemical probes selective for non-B DNA structures provides evidence that the MT-I R/Y sequence adopts one or more H-DNA structures. We also investigated this sequence to determine if it can influence transcriptional regulation. Promoter/reporter constructs were prepared in which the MT-I R/Y sequence was positioned in either orientation upstream of either the MT-I or HSV-TK promoters. Promoter/reporter activities were evaluated by transient transfection assays using mouse NIH3T3 cells. The MT-I R/Y sequence displayed no detectable activity as a cis -acting transcriptional regulatory element. These results demonstrate that although the MT-I R/Y sequence is able to adopt a non-B DNA structure under certain in vitro conditions, there is no evidence that this sequence plays a significant role in transcriptional regulation.

3T3 Cells↗

PubMed1998

Electrostatic effects in DNA bending by GCN4 mutants.

DNA architecture has been shown to be important for cellular processes such as activation of transcription, recombination, and replication. Many proteins reconfigure the shape of duplex DNA upon binding. Previous experiments have shown that some members of the eukaryotic bZIP family of DNA binding proteins appear to bend DNA, while others do not. We are exploring the role of electrostatic effects in DNA bending by bZIP proteins. The yeast bZIP transcription factor GCN4 does not induce DNA bending in vitro. Previously we substituted basic residues for three neutral amino acids in GCN4 to produce a GCN4 derivative that bends DNA by approximately 15 degrees. This result is consistent with a model of induced DNA bending wherein excess positive charge in proximity to one face of the double helix neutralizes local phosphate diester anions resulting in a laterally-asymmetric charge distribution along the DNA. Such an unbalanced charge distribution can result in collapse of the DNA toward the neutralized surface. We now present a more comprehensive analysis of electrostatic effects in DNA bending by GCN4 derivatives. It is shown that the direction and extent of DNA bending by these derivatives are a linear function of the charges of the amino acids adjacent to the basic domain of the protein. This relation holds over the charge range +6 (16 degrees bend toward the minor groove) to -6 (25 degrees bend toward the major groove).

Amino Acid Sequence↗

PubMed1998

Mechanisms of DNA bending.

Genome packaging and gene regulation require DNA bending. Recent developments in the elucidation of the mechanisms involved in DNA bending include new X-ray structures (most notably that of the mammalian nucleosome) wherein DNA is bent, controversy surrounding interpretation of DNA-bending experiments with basic-leucine zipper proteins, studies of electrostatic effects in DNA bending, and the design of artificial DNA-bending ligands.

Animals↗

PubMed1997

Role of asymmetric phosphate neutralization in DNA bending by PU.1.

The PU.1 transcription factor is a member of the Ets family of DNA binding proteins. PU.1 binds to DNA via a loop-helix-loop domain and functions in the differentiation of hematopoietic cells. The structure of a PU.1-DNA complex was recently reported (Kodandapani, R., Pio, F., Ni, C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R., and Ely, K. (1996) Nature 380, 456-460). The DNA in this complex is deformed by 8 degrees as it curves around the protein. The pattern of electrostatic contacts between PU.1, and its DNA binding site suggests that laterally asymmetric phosphate neutralization accompanies PU.1 binding. Because of our previous studies showing that such neutralization can induce bending in naked DNA, we have explored the effect of phosphate neutralization by substituting neutral methylphosphonate internucleoside linkages at relevant positions within DNA containing the PU.1 binding sequence. Consistent with the prediction that DNA will collapse toward its partially neutralized surface, DNA neutralized at seven positions to simulate PU.1 binding is observed to bend by 28 degrees . The directions of DNA curvature are slightly different in the co-crystal versus the partially neutralized duplexes. The electrostatic component of the binding energy appears more than enough to account for the DNA bending observed in the PU.1-DNA complex.

Binding Sites↗

PubMed1997

DNA bending by GCN4 mutants bearing cationic residues.

Transcription activation is thought to require DNA bending to promote the interaction of upstream activators and the basal transcription machinery. Previous experiments have shown that some members of the bZIP family of DNA binding proteins bend DNA, while others do not. We are exploring the possibility that electrostatic effects play a role in these differences. The yeast bZIP transcription factor GCN4 does not induce DNA bending in vitro. Substitution of basic residues for three neutral amino acids of GCN4 confers the ability to bend DNA. This result is consistent with a model of induced DNA bending wherein excess positive charge in proximity to one face of the double helix neutralizes local phosphate diester anions resulting in a laterally asymmetric charge distribution along the DNA. Previous data suggest that such an unbalanced charge distribution results in collapse of the DNA toward the neutralized surface. Interpretations of the present data are discussed. Our result supports the hypothesis that electrostatic interactions can play a key role in DNA bending by bZIP proteins.

Base Sequence↗

PubMed1997

Effects of neutralization pattern and stereochemistry on DNA bending by methylphosphonate substitutions.

Asymmetric phosphate neutralization has been hypothesized to play a role in DNA bending by proteins. Neutralization is thought to involve salt bridges between the negatively charged phosphate backbone of duplex DNA and the cationic amino acids of an approaching protein. According to this model, the resulting unbalanced charge distribution along the duplex DNA induces the double helix to collapse toward the neutralized surface. Previous work has confirmed that DNA bending is induced by the asymmetric incorporation of racemic methylphosphonate linkages creating a neutral region on one face of duplex DNA. Neutralization was accomplished by substitution of three consecutive phosphodiesters on each strand, arranged across one minor groove of the DNA (a total of six neutralized phosphates). We now measure DNA bending induced by a more diffuse patch of neutralization (alternating neutralized and anionic phosphates) and explore the effect of methylphosphonate stereochemistry. DNA duplexes with patches of alternating methylphosphonate and phosphodiester linkages are less bent than DNAs wherein consecutive phosphates are neutralized. Furthermore, duplexes neutralized by incorporation of pure (RP)-methylphosphonate isomers are bent approximately 30% less than duplexes neutralized by racemic methylphosphonates.

Base Sequence↗

PubMed1997

Quantitating oligonucleotide affinities for duplex DNA: footprinting vs electrophoretic mobility shift assays.

Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. Quantitative DNa-seI footprinting assays (QDFA) offer a rigorous technique for this purpose. Electrophoretic mobility shift assays (EMSA) have proven to be simpler and more rapid. Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by QDFA. We show that QDFA and EMSA techniques provide Kd estimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mM). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods.

DNA↗

PubMed1996

A potential H-DNA element in the MUC1 promoter does not influence transcription.

A purine/pyrimidine mirror repeat element (M-PMR3) in the MUC1 promoter has been shown to form H-DNA under in vitro conditions. We investigated this element for biological function in the regulation of transcription of this gene. Chloramphenicol acetyltransferase reporter-promoter constructs were prepared in which the mirror repeat element (PMR3) was intact, deleted, or modified, and their activities were evaluated by transient transfection assays into the cell lines Capan-2, PANC1, and HT-29. Deletion or modification of M-PMR3 increased expression of chloramphenicol acetyltransferase activity in MUC1-expressing cells; however, a role for an H-DNA structure in this activity was not supported by the results.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1996

DNA bending by hexamethylene-tethered ammonium ions.

DNA is bent when complexed with certain proteins. We are exploring the hypothesis that asymmetric neutralization of phosphate charges will cause the DNA double helix to collapse toward the neutralized face. We have previously shown that DNA spontaneously bends toward one face of the double helix when it is partially substituted with neutral methylphosphonate linkages. We have now synthesized DNA duplexes in which cations are tethered by hexamethylene chains near specific phosphates. Electrophoretic phasing experiments demonstrate that tethering six ammonium ions on one helical face causes DNA to bend by approximately 5 degrees toward that face, in qualitative agreement with predictions. Ion pairing between tethered cations and DNA phosphates provides a new model for simulating the electrostatic consequences of phosphate neutralization by proteins.

Base Sequence↗

PubMed1996

Potential for H-DNA in the human MUC1 mucin gene promoter.

Similar imperfect purine/pyrimidine mirror repeat (PMR) elements have previously been identified upstream of the human MUC1 mucin and CFTR genes. These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH. We now present a detailed characterization of the non-B DNA structure responsible for S1 nuclease sensitivity upstream of the MUC1 gene. A approximately 90-base pair (bp) DNA fragment containing a 32-bp PMR element termed M-PMR3 was subcloned into a recombinant vector. This fragment conferred S1 nuclease sensitivity on the resulting supercoiled plasmid. High resolution mapping of sites reactive to S1 and P1 nucleases demonstrates that cleavage occurs within the M-PMR3 element. High resolution mapping with chemical agents selective for non-B DNA provides evidence that M-PMR3 adopts an H-DNA structure (intramolecular triple helix) in the less common H-y5 isomer at low pH. This result is observed in the presence or absence of Mg2+. Mutation of the native M-PMR3 element to create perfect homopurine/homopyrimidine mirror symmetry alters the preferred folding to the more common H-y3 triplex DNA isomer. These results demonstrate that imperfections in mirror symmetry can alter the relative stabilities of different H-DNA isomers.

Base Sequence↗

PubMed1996

Selection of RNAs that bind to duplex DNA at neutral pH.

RNA that are capable of binding duplex DNA in a site-specific manner have potential applications in gene therapy strategies. Such RNAs might be targeted to DNA sequences in a gene promoter and prevent initiation of transcription by occluding transcription factors and/or RNA polymerases. RNA oligonucleotides that bind homopurine/homopyrimidine DNA sequences by forming triple-helical complexes involving T.A.T and C+.G.C base-triplets can be rationally designed. However, the formation of such pyrimidine motif triple helices typically requires mildly acidic conditions. In addition, the proper oligonucleotide sequence must be optimally presented within a longer RNA transcript if it is to be synthesized in vivo. To address these issues, RNAs were selected from pools of random sequences for binding to a homopurine/homopyrimidine DNA sequence. RNAs selected for binding the duplex DNA target between pH 6.5 and pH 7.4 were characterized by sequence analysis and binding studies. All RNAs isolated by selection and amplification were found to contain a pyrimidine recognition sequence for binding the duplex DNA target via conventional triple helix formation. The selected approximately 85 nt RNAs have dissociation constants that approach, but do not surpass, the binding affinity of a 21 nt RNA oligonucleotide that binds the DNA target sequence by forming a canonical triple helix. The presence of a pyrimidine recognition sequence within a longer RNA transcript is not sufficient for high affinity. Experimental data and secondary structure predictions suggest that the context of the pyrimidine recognition sequence within selected RNAs is a very important determinant of DNA binding affinity. These studies provide insight into the development of RNA transcripts that may function as gene-specific repressors by forming triple helices with DNA in vivo.

Base Sequence↗

PubMed1996

Binding of DNA oligonucleotides to sequences in the promoter of the human bc1-2 gene.

Duplex DNA recognition by oligonucleotide-directed triple helix formation is being explored as a highly specific approach to artificial gene repression. We have identified two potential triplex target sequences in the promoter of the human bcl-2 gene, whose product inhibits apoptosis. Oligonucleotides designed to bind these target sequences were tested for their binding affinities and specificities under pseudo-physiological conditions. Electrophoretic mobility shift and dimethyl sulfate footprinting assays demonstrated that an oligonucleotide designed for simultaneous recognition of homopurine domains on alternate duplex DNA strands had the highest affinity of any oligonucleotide tested. Modifications to render this oligonucleotide nuclease-resistant did not reduce its binding affinity or specificity. In additional studies under various pH conditions, pyrimidine motif complexes at these target sequences were found to be stable at pH 8.0, despite the presumed requirement for protonation of oligonucleotide cytidines. In contrast, purine motif complexes, typically considered to be pH independent, were highly destabilized at decreasing pH values. These results indicate that a natural sequence in the human bcl-2 promoter can form a stable triplex with a synthetic oligonucleotide under pseudo-physiological conditions, and suggest that triple helix formation might provide an approach to the artificial repression of bcl-2 transcription.

Base Sequence↗

PubMed1996

DNA bending by a phantom protein.

BACKGROUND: Despite its stiffness, duplex DNA is extensively bent and folded during packaging and gene expression in biological systems. Modulation of the electrostatic repulsion between phosphates in the DNA backbone may be important in the bending of DNA by proteins. Here, we analyze the shape of DNA molecules that have been modified chemically to mimic the electrostatic consequences of a bound protein. RESULTS: We have simulated salt bridges between DNA phosphates and cationic amino acid sidechains of a phantom protein by tethering ammonium cations to one face of the DNA helix. Tethered ammonium cations, but not neutral acetylated controls, induce DNA to bend toward its neutralized surface. CONCLUSIONS: The shape of DNA molecules bearing a laterally-asymmetric distribution of tethered cations agrees qualitatively with theoretical predictions and with results previously obtained using neutral phosphate analogs. These data suggest principles that might be applied to the design of artificial DNA-bending proteins.

Base Sequence↗

PubMed1996

Prospects for the therapeutic use of antigene oligonucleotides.

An outgrowth of classic nucleic acid interaction studies, oligonucleotide-directed triple helix formation is a unique method for creating highly specific chemical ligands that recognize and bind to particular sequences of duplex DNA. Under permissive conditions, these oligonucleotide-based compounds can approach or exceed the binding affinity and sequence specificity of natural DNA-binding proteins. Triple helix recognition has been found to be useful in certain cell-free applications including precise chromosome fragmentation. It has been proposed that such oligonucleotides could also form the basis for gene-targeted (antigene) drugs that might repress transcription from undesired genes in living cells. However, current strategies for oligonucleotide-directed triple helix formation suffer from important constraints involving requirements for stabilizing binding conditions, restrictions on permitted target sequences, and inefficient nuclear delivery of oligonucleotides. Implementation of oligonucleotide-directed triple helix formation as a viable approach to cancer therapy must therefore await clever solutions to a series of fascinating problems.

Animals↗

PubMed1995

Overcoming potassium-mediated triplex inhibition.

Sequence-specific duplex DNA recognition by oligonucleotide-directed triple helix formation is a possible approach to in vivo gene inhibition. However, triple helix formation involving guanine-rich oligonucleotides is inhibited by physiological ions, particularly K+, most likely due to oligonucleotide aggregation via guanine quartets. Three oligodeoxynucleotide (ODN) derivatives were tested for their ability to resist guanine quartet-mediated aggregation, yet form stable triplexes. Electrophoretic mobility shift and dimethyl sulfate footprinting assays were used to analyze the formation of triplexes involving these oligonucleotide derivatives. In the absence of K+, all ODNs had similar binding affinities for the duplex target. Triplexes involving a 14mer ODN derivative containing 7-deazaxanthine substituted for three thymine bases or an 18mer ODN containing two additional thymines on both the 5' and 3' termini were abolished by 50 mM K+. Remarkably, triplexes involving an ODN derivative containing four 6-thioguanine bases substituted for guanine resisted K+ inhibition up to 200 mM. We hypothesize that the increased radius and decreased electronegativity of sulfur in the 6-position of guanine destabilize potential guanine quartets. These results improve the prospects for creating ODNS that might serve as specific and efficient gene repressors in vivo.

Animals↗

PubMed1995

Recognition of duplex DNA by RNA polynucleotides.

We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids. Homopyrimidine RNA oligonucleotides bind to duplex DNA at homopurine/homopyrimidine sequences under slightly acidic conditions. Recognition is sequence-specific, involving rU.dA.dT and rC+.dG.dC base triplets. Affinities were determined for folded polymeric RNAs (ca. 100-200 nt) containing 0, 1 or 3 copies of a 21 nt RNA sequence that binds duplex DNA by triple helix formation. When this recognition sequence was inserted into the larger folded RNAs, micromolar concentrations of the resulting RNA ligands bound a duplex DNA target at pH 5. However, these binding affinities were at least 20-fold lower than the affinity of an RNA oligonucleotide containing only the recognition sequence. Enzymatic probing of folded RNAs suggests that reduced affinity arises from unfavorable electrostatic, structural and topological considerations. The affinity of a polymeric RNA with three copies of the recognition sequence was greater than that of a polymeric RNA with a single copy of the sequence. This affinity difference ranged from 2.6- to 13-fold, depending on pH. Binding of duplex DNA by polymeric RNA might be improved by optimizing the RNA structure to efficiently present the recognition sequence.

Base Sequence↗

PubMed1995

Competitive triplex/quadruplex equilibria involving guanine-rich oligonucleotides.

Oligonucleotide-directed triple helix formation in the purine motif involves the binding of guanine-rich oligonucleotides to duplex DNA. Although this approach has been proposed for in vivo gene inhibition, triple helix formation by guanine-rich oligonucleotides is severely inhibited by physiological concentrations of certain monovalent cations (M+), especially K+. To clarify the mechanism of this inhibition, electrophoretic gel mobility shift titrations were performed to analyze the formation and stability of a purine motif triple helix in the presence of M+ and to monitor oligonucleotide aggregation under these conditions. M+ inhibition of triplex formation exhibited a concentration and ionic radius dependence that correlates with the ability of M+ to stabilize guanine quartet structures. In the presence of inhibitory [M+], guanine-rich oligonucleotides formed aggregates having characteristics consistent with the involvement of guanine quartets. The inhibitory effects of K+ on triplex formation could not be reversed by addition of the physiological polyamines spermidine3+ or spermine4+. M+ reduced the equilibrium concentration of the triplex primarily by decreasing the rate of triplex formation, but M+ also caused a detectable increase in the rate of triplex dissociation. Together, these results suggest that triplex inhibition under physiological ionic conditions is caused by competing equilibria wherein guanine-rich oligonucleotides form aggregates involving guanine quartets. Approaches to destabilizing aggregates of guanine-rich oligonucleotides under physiological conditions will be required before in vivo applications can be realistically considered.

Base Sequence↗