1,721,109 research outputs found
Probing the conformations of eight cloned-DNA dodecamers; CGCGAATTCGCG, CGCGTTAACGCG, CGCGTATACGCG, CGCGATATCGCG, CGCAAATTTGCG, CGCTTTAAAGCG, CGCGGATCCGCG and CGCGGTACCGCG
No full textThe self complementary DNA dodecamers d(CGCGAATTCGCG), d(CGCGTTAACGCG), d(CGCGTATACGCG), d(CGCGATATcGcG), d(CGCAAATTTGCG), d(CGCTTTAAAGCG), d(CGCGGATCCGCG) and d(CGCGGTACCGCG) have been cloned into the Smal site of plasmid pUC19. Radiolabelled polylinker fragments containing these inserts have been digested with nucleases and chemical agents, probing the structure of the central AT base pairs. The sequences AATT and AAATTT are relatively resistant to digestion by DNase I, micrococcal nuclease and hydroxyl radicals, consistent with the suggestion that they possess a narrow minor groove. Nuclease digestion of TTAA is much more even, and comparable to that at mixed sequence DNA. TpA steps in ATAT, TATA and GTAC are cut less well by DNAse I than In TTAA. DNasel cleavage of surrounding bases, especially CpG is strongly influenced by the nature of the central sequence.</p
Formation of DNA triple helices incorporating blocks of G·GC and T·AT triplets using short acridine-linked oligonucleotides
No full textWe have used DNase I footprinting to assess triple helix formation at target sites containing the sequences A6G6·C6T6 and G6A6·T6C6. These sequences can be recognized by the acridine-linked oligopyrimidines Acr-T5C5 and Acr-C5T5 respectively at low pH, using well-characterised T·AT and C+·GC triplets. At pH 7.5 A6G6·C6T6 is specifically bound by Acr-G5T5, utilising G·GC and T·AT triplets in which the third strand runs antiparallel to the purine strand of the duplex. This interaction requires the presence of magnesium ions. No interaction was detected with Acr-T5G5, an oligonucleotide designed to form parallel G·GC and T·AT triplets. In contrast neither Acr-T5G5 nor Acr-G5T5 produced DNase I footprints with the target sequence G6A6·T6C6. These results suggest that, in an antiparallel R·RY triple helix, the T·AT triplet is weaker than the G·GC triplet. We find no evidence for the formation of structures containing parallel G·GC triplets.</p
Wrapping of genomic polydA.polydT tracts around nucleosome core particles
No full textFive human clones containing genomic regions of polydA have been isolated by their ability to form intermolecular triple helices with agarose cross-linked polyU. All of these clones contain Alu repetitive DNA sequences. End-labelled DNA fragments containing these sequences have been successfully reconstituted onto nucleosome core particles by salt exchange. The structure of these has been examined by digesting with DNase I, hydroxyl radicals or diethylpyrocarbonate. DNase I cleavage of the polydA tracts is poor in the free DNA but is markedly enhanced at certain positions when complexed with nucleosome cores. Phased digestion patterns are observed which continue through the (A)n blocks and reveal an average helical periodicity of about 10 base pairs. The distance between adjacent maxima varies between 8-12 base pairs, suggesting that the exact helical repeat is not necessarily constant. One fragment containing the sequence (TA)11T34 reveals a 12 base pair repeat within the (AT)n region. A pUC19 polylinker fragment containing a block of A69.T69 cloned into the Smal site could also be reconstituted onto nucleosome cores and reveals the same phased DNasel digestion pattern. The DNase I cleavage pattern is not identical at each of the maxima, suggesting that the structural distortions imposed by the core particles are not constant along the DNA.</p
Footprinting studies of DNA‐sequence recognition by nogalamycin
No full textWe have studied the DNA sequence binding preference of the antitumour antibiotic nogalamycin by DNase‐I footprinting using a variety of DNA fragments. The DNA fragments were obtained by cloning synthetic oligonucleotides into longer DNA fragments and were designed to contain isolated ligand‐binding sites surrounded by repetitive sequences such as (A)n· (T)n and (AT)n. Within regions of (A)n· (T)n, clear footprints are observed with low concentrations of nogalamycin (< 5 μM), with apparent binding affinities for tetranucleotide sequences which decrease in the order TGCA > AGCT = ACGT > TCGA. In contrast, within regions of (AT)n, the ligand binds best to AGCT; binding to TCGA and TGCA is no stronger than to alternating AT. Within (ATT)n, the preference is for ACGT > TCGA. Although each of these binding sites contains all four base pairs, there is no apparent consensus sequence, suggesting that the selectivity is affected by local DNA dynamic and structural effects. At higher drug concentrations (> 25 μM), nogalamycin prevents DNAse‐I cleavage of (AT)n but shows no interaction with regions of (AC)n· (GT)n. Regions of (A)n· (T)n, which are poorly cut by DNase I, show enhanced rates of cleavage in the presence of low concentrations of nogalamycin, but are protected from cleavage at higher concentrations. We suggest that this arises because drug binding to adjacent regions distorts the DNA to a structure which is more readily cut by the enzyme and which is better able to bind further ligand molecules.</p
Secondary (non-GpC) binding sites for actinomycin on DNA
No full textActinomycin D has long been known to bind selectively to the dinucleotide step GpC. We have investigated its ability to bind to other non-canonical sequences using a series of synthetic DNA fragments. DNase I footprinting experiments reveal that actinomycin can also bind well to GG (CC). Binding to this sequence and the canonical GC site is potentiated by flanking regions of (GT)n·(AC)n Weaker but specific binding to GT and AC is also evident and appears to be cooperative.</p
The stability of intramolecular DNA quadruplexes with extended loops forming inter- and intra-loop duplexes
No full textInteraction of echinomycin with A<sub>n</sub>.T<sub>n</sub>. and (AT)<sub>n</sub> regions flanking its CG binding site
No full textWe have prepared DNA fragments containing the sequences A15CGT15,T15and T(AT)8CG(AT)15 cloned within the Smal site of the pUC19 poly I Inker. These have been used as substrates in footprinting experiments with DNase I and diethylpyrocarbonate probing the effects of echinomycin, binding to the central CG, on the structure of the surrounding sequences. No clear DNase I footprints are seen with T15CGA15 though alterations in the nuclease susceptibility of surrounding regions suggest that the ligand is binding, albeit weakly at this site. All the other fragments show the expected footprints around the CG site. Regions of An and Tn are rendered much more reactive to DNase I and adenines on the 3′-side of the CG become hyperreactive to diethylpyrocarbonate. Regions of alternating AT show unusual changes in the presence of the ligand. At low concentrations (5μM) cleavage of TpA is enhanced, whereas at higher concentrations a cleavage pattern with a four base pair repeat is evident. A similar pattern is seen with micrococcal nuclease. Modification by diethylpyrocarbonate is strongest at alternate adenines which are staggered in the 5′-direction across the two strands. We interpret these changes by suggesting secondary drug binding within regions of alternating AT, possibly to the dinucleotide ApT. DNase I footprinting experiments performed at 4°C revealed neither enhancements nor footprints for flanking regions of homopolymeric A and T suggesting that the conformational changes are a necessary consequence of drug binding.</p
Kinetic studies on the formation of acridine-linked DNA triple helices
No full textAbstractWe have used DNase I footprinting to measure the rate of intermolecular triple helix formation at the target sites A6G6 · C6T6 and G6A6 · T6C6 with the acridine-linked oligonucleotides Acr-T5C5, and Acr-C5T5 respectively. Under pseudo first-order reaction conditions we find that the reactions are slow, with half-lives of several minutes. The rates are dependent on the concentration of the third strand oligonucleotide and yield bimolecular association rate constants of 300 M−1 · s−1 for Acr-T5C5 binding to A6G6 · C6T6 and 2000 M−1 · s−1 for the interaction of Acr-C5T5 with G6A6 · T6C6
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