Detection of CAG repeat DNA sequences by pyrene-functionalized pyrrole-imidazole polyamides

Five N-methylpyrrole-N-methylimidazole (Py-Im) polyamides possessing a fluorescent pyrene were synthesized by Fmoc solid-phase synthesis using Py/Im monomers and pyrenylbutyl-pyrrole monomer compound 9. The steady state fluorescence of conjugates 1-5 was examined in the presence and absence of (CAG)(12)-containing oligodeoxynucleotides (ODNs) 1 and 2. Of the conjugates, conjugate 1 showed no background emission around 470 nm in the absence of ODNs, and a clear increase of emission at 475 nm was observed upon addition of ODNs 1 and 2. The emission of conjugate 1 at 475 nm increased linearly with the concentration of ODN and the number of CAG repeats. The results indicate that conjugate 1 efficiently forms a pyrene excimer upon binding in the minor groove of DNA.     

Evolutionary origin of expandable G-C-rich triplet repeat DNA sequences

A model explaining properties exhibited by fragile-X DNA systems arises from observations that time-dependent base substitutions are expressed at G-C sites but not at A–T sites (Biochem. Genet.32:383, 1994). [CGG]_n sequences are classified as most sensitive to evolutionary base substitution processes involving time-dependent populating of G-C sites with enol-imine states having enhanced stability. Increased density of these states in oocyte DNA would introduce a ground-state collapse double-helix of reduced energy that would inhibit strand separation by the replicase. Evolutionarily altered G in CGG triplets allows CGG to be transcribed as CTG, an initiation codon. And this will cause reinitiation of DNA synthesis, thereby adding additional CGG units to the collapsed double helix. This situation would not occur in slower-evolving male haploid DNA that replicates frequently.     

Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides

The ability of DNA-binding proteins to recognize their cognate sites in chromatin is restricted by the structure and dynamics of nucleosomal DNA, and by the translational and rotational positioning of the histone octamer. Here, we use six different pyrrole-imidazole polyamides as sequence-specific molecular probes for DNA accessibility in nucleosomes. We show that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible and that nucleosomes remain fully folded upon ligand binding. Polyamides only failed to bind where sites are completely blocked by interactions with the histone octamer. Removal of the amino-terminal tails of either histone H3 or histone H4 allowed these polyamides to bind. These results demonstrate that much of the DNA in the nucleosome is freely accessible for molecular recognition in the minor groove, and also support a role for the amino-terminal tails of H3 and H4 in modulating accessibility of nucleosomal DNA.     

Targeted derepression of the human immunodeficiency virus type 1 long terminal repeat by pyrrole-imidazole polyamides

The host factor LSF represses the human immunodeficiency virus type 1 long terminal repeat (LTR) by mediating recruitment of histone deacetylase. We show that pyrrole-imidazole polyamides targeted to the LTR can specifically block LSF binding both in vitro and within cells via direct access to chromatin, resulting in increased LTR expression.     

Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.     

Genetic instabilities of triplet repeat sequences by recombination

The expansion of triplet repeat sequences is an initial step in the disease etiology of a number of hereditary neurological disorders in humans. Diseases such as myotonic dystrophy, Huntington's, several spinocerebellar ataxias, fragile X syndrome, and Friedreich's ataxia are caused by the expansions of CTG.CAG, CGG.CCG, or GAA.TTC repeats. The mechanisms of the expansion process have been investigated intensely in E. coli, yeast, transgenic mice, mammalian cell culture, and in human clinical cases. Whereas studies from 1994-1999 have implicated DNA replication and repair at the paused synthesis sites due to the unusual conformations of the triplet repeat sequences, recent work has shown that homologous recombination (gene conversion) is a powerful mechanism for generating massive expansions, in addition to, or in concert with, replication and repair.     

Recognition of the DNA minor groove by pyrrole-imidazole polyamides: comparison of desmethyl- and N-methylpyrrole

Polyamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp) are synthetic ligands that recognize predetermined DNA sequences with affinities and specificities comparable to many DNA-binding proteins. As derivatives of the natural products distamycin and netropsin, Py/Im/Hp polyamides have retained the N-methyl substituent, although structural studies of polyamide:DNA complexes have not revealed an obvious function for the N-methyl. In order to assess the role of the N-methyl moiety in polyamide:DNA recognition, a new monomer, desmethylpyrrole (Ds), where the N-methyl moiety has been replaced with hydrogen, was incorporated into an eight-ring hairpin polyamide by solid-phase synthesis. MPE footprinting, affinity cleavage, and quantitative DNase I footprinting revealed that replacement of each Py residue with Ds resulted in identical binding site size and orientation and similar binding affinity for the six-base-pair (bp) target DNA sequence. Remarkably, the Ds-containing polyamide exhibited an 8-fold loss in specificity for the match site versus a mismatched DNA site, relative to the all-Py parent. Polyamides with Ds exhibit increased water solubility, which may alter the cell membrane permeability properties of the polyamide. The addition of Ds to the repertoire of available monomers may prove useful as polyamides are applied to gene regulation in vivo. However, the benefits of Ds incorporation must be balanced with a potential loss in specificity.     

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli

The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats). The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.     

Unusual capillary electrophoretic behavior of triplet repeat DNA

We investigated the electrophoretic behavior of triplet repeat DNA fragments by capillary electrophoresis and found triplet repeat DNA fragments showed unusual mobilities compared with those of commercially available DNA molecular marker. The electrophoretic data are analyzed by means of Ogston model and the mechanism of a change in mobility of triplet repeat DNA is discussed. The unusual mobilities are caused by the characteristic higher-order structure formed by GC-rich triplet repeat DNA.     

Localizing triplet periodicity in DNA and cDNA sequences

Background  

The protein-coding regions (coding exons) of a DNA sequence exhibit a triplet periodicity (TP) due to fact that coding exons contain a series of three nucleotide codons that encode specific amino acid residues. Such periodicity is usually not observed in introns and intergenic regions. If a DNA sequence is divided into small segments and a Fourier Transform is applied on each segment, a strong peak at frequency 1/3 is typically observed in the Fourier spectrum of coding segments, but not in non-coding regions. This property has been used in identifying the locations of protein-coding genes in unannotated sequence. The method is fast and requires no training. However, the need to compute the Fourier Transform across a segment (window) of arbitrary size affects the accuracy with which one can localize TP boundaries. Here, we report a technique that provides higher-resolution identification of these boundaries, and use the technique to explore the biological correlates of TP regions in the genome of the model organism C. elegans.     

Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences

The influence of negative superhelical density on the genetic instabilities of long GAA.TTC, CGG.CCG, and CTG.CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (-sigma). All three strategies revealed that higher -sigma in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of -sigma stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slippage of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the -sigma of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases.     

Sequence specific recognition of DNA by tailor-made hairpin conjugates of achiral seco-cyclopropaneindoline-2-benzofurancarboxamide and pyrrole-imidazole polyamides

Hairpin conjugates of achiral seco-cyclopropaneindoline-2-benzofurancarboxamide (achiral seco-CI-Bf) and three diamides (ImPy 1, PyIm 2, and PyPy 3, where Py is pyrrole, and Im is imidazole), linked by a gamma-aminobutyrate group, were synthesized. The sequence-specific covalent alkylation of the achiral CI moiety with adenine-N3 in the minor groove was ascertained by thermally induced DNA cleavage experiments. The results provide evidence that hairpin conjugates of achiral seco-CI-Bf-gamma-polyamides could be tailored to target specific DNA sequences according to a set of general rules: the achiral CI moiety selectively reacts with adenine-N3, a stacked pair of imidazole/benzofuran prefers a G/C base pair, and a pyrrole/benzofuran prefers an A/T or T/A base pair. Models for the binding of hairpin conjugates 1-3 with sequences 5'-TCA(888)G-3', 5'-CAA(857)C-3', and 5'-TTA(843)C-3' are proposed.     

Detection of specific DNA sequences using antibodies recognizing UV-labelled DNA.

This non-isotopic method for detection of nucleic acids is based on the in situ labelling of the nucleic acid by exposure to UV-irradiation. The different UV-induced photoproducts, mainly of the thymidine dimer type, are recognized by purified rabbit antibodies specific to the lesions introduced. The UV-labelled nucleic acids can then be visualized by conventional immunostaining procedures. A major advantage of the technique is the low cost and the ease by which the DNA is specifically labelled. The purified rabbit antibodies were shown to be specific for UV-irradiated DNA, and the method was applied for detection of specific DNA sequences hybridized to homologous target DNA on membrane support. We believe that the sensitivity of the method can be improved, and the significance of using different UV-doses, immunostaining methods and membrane types is discussed.     

The structure of 4-way DNA junctions: specific binding of bis-intercalators with rigid linkers.

During replication and recombination, two DNA duplexes lie side by side. We have developed reagents that might be used to probe structure during these critical processes; they contain two intercalating groups connected by a rigid linker that forces those groups to point in opposite directions. If their stereochemistry proves appropriate, such structure-specific agents should intercalate specifically into adjacent duplexes in the Y- and X-shaped structures (i.e. 3- and 4-way junctions, now known as 3H and 4H junctions) found at replication and recombination sites. We prepared DNA structures in which four duplexes were arranged in all possible combinations around 2- and 4-way junctions and then probed the accessibility to DNase I of all their phosphodiester bonds. In the absence of any bis-intercalators, 7-9 nucleotides (nt) in each of the strands in 4-way junctions were protected from attack; protected regions were significantly offset to the 3' side of the junction in continuous strands, but only slightly offset, if at all, in exchanging strands. All the intercalators decreased accessibility throughout the structure, but none did so at specific points in the two adjacent arms of 4-way junctions. However, one bis-intercalator--but not its sister with a shorter linker--strikingly increased access to a particular CpT bond that lay 9 nt away from the centre of some 4-way junctions without reducing access to neighbouring bonds. Binding was both sequence and structure specific, and depended on complementary stereochemistry between bis-intercalator and junction.