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K R Fox

Publications and source records attributed to K R Fox.

At least 109 records · Page 6Linked to original sources
PubMed1992

Probing the conformations of eight cloned DNA dodecamers; CGCGAATTCGCG, CGCGTTAACGCG, CGCGTATACGCG, CGCGATATCGCG, CGCAAATTTGCG, CGCTTTAAAGCG, CGCGGATCCGCG and CGCGGTACCGCG.

The 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.

Base Sequence↗
PubMed1992

Footprinting studies of DNA-sequence recognition by nogalamycin.

We 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 microM), 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 microM), 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.

Base Sequence↗
PubMed1992

Secondary (non-GpC) binding sites for actinomycin on DNA.

Actinomycin 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.

Base Sequence↗
PubMed1992

Bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM binds to the dinucleotide TpA.

The binding of [N-MeCys3,N-MeCys7]TANDEM has been examined by DNase I footprinting and diethyl pyrocarbonate modification of several synthetic DNA fragments containing AT-rich regions. DNase I footprinting reveals that at low concentrations the ligand binds preferentially to the center of (AT)n regions. A fragment containing the tetranucleotide AATT was unaffected by the ligand. Diethyl pyrocarbonate modification of several fragments containing blocks of (AT)n revealed a pattern in which alternate adenines were rendered more reactive in the presence of the ligand. These reactive adenines were staggered across the two DNA strands in the 3'-direction, consistent with ligand binding to the dinucleotide TpA. In sequences of the type (TAA)n.(TTA)n, binding of [N-MeCys3,N-MeCys7]TANDEM resulted in strong modification of the second adenine in the sequence TAA, i.e., the base on the 3'-side of the ligand binding site. Data for binding to (AT)n are best explained by suggesting that the adenines sandwiched between the quinoxaline chromophores are rendered most reactive to diethyl pyrocarbonate.

Base Sequence↗
PubMed1992

Interaction of bleomycin with a bent DNA fragment.

The interaction of bleomycin with a kinetoplast DNA fragment has been examined using various footprinting techniques. This DNA adopts a bent structure and displays an unusually low gel mobility on account of its phased runs of adenines. The bleomycin-cobalt complex increases the mobility of this DNA fragment, in contrast with other DNAs which show a decreased rate of gel migration, suggesting that the antibiotic removes DNA bending, possibly via an unwinding mechanism. Removal of the bending is confirmed by hydroxy-radical footprinting which produces a more even ladder of bands in the presence of the ligand. Cleavage by bleomycin is at the sequence G-pyrimidine, though not all such sites are affected to the same extent and some cutting is found at GA and GG. DNase I footprinting confirms the antibiotic-binding sites but reveals that some strong cleavage sites do not yield footprints. Bleomycin renders adenines on the 3' side of its cleavage sites (GT, GC and GA) hyper-reactive to diethyl pyrocarbonate.

Base Sequence↗
PubMed1992

Wrapping of genomic polydA.polydT tracts around nucleosome core particles.

Five 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 DNaseI 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.

Base Sequence↗
PubMed1992

Methyl green and its analogues bind selectively to AT-rich regions of native DNA.

Methyl green has long been used as a DNA stain in histochemistry. The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting. At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T. At higher concentrations the dyes bound to almost all available DNA sites. Models, with and without intercalation are discussed to account for the specific binding.

Base Sequence↗
PubMed1991

Interaction of echinomycin with An.Tn. and (AT)n regions flanking its CG binding site.

We have prepared DNA fragments containing the sequences A15CGT15, T15CGA15 and T(AT)8CG(AT)15 cloned within the SmaI site of the pUC19 polylinker. 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 (5 microM) 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 degrees C revealed neither enhancements nor footprints for flanking regions of homopolymeric A and T suggesting that the conformational changes are necessary consequence of drug binding.

Amino Acid Sequence↗
PubMed1991

Echinomycin binding to alternating AT.

We have studied the binding of echinomycin to DNA fragments containing GC-rich regions flanked by blocks of alternating AT by DNase I footprinting and diethylpyrocarbonate modification. Regions of alternating AT flanking the sequences CCCG, CCGC, CGGC and GG show a four base pair DNase I cleavage pattern and reaction of alternate adenines with diethylpyrocarbonate. This pattern is strongest when the AT-block is immediately adjacent to the CpG ligand binding site. We explain these phenomena by suggesting that echinomycin binds to the dinucleotide step ApT in a cooperative fashion. The cooperative effects can be transmitted through the dinucleotide step GC but not CC or AA. No such repetitive patterns are seen with surrounding regions of (ATT).(AAT). Evidence is presented for secondary drug binding sites at CpC and TpG with weaker interaction at the CpG site within the hexanucleotide TTCGAA.

Base Sequence↗
PubMed1991

Effects of the antitumor antibiotic mithramycin on the structure of repetitive DNA regions adjacent to its GC-rich binding site.

Regions of An.Tn, (GA)n.(TC)n, and (GT)n.(AC)n have been cloned into the SmaI (CCC/GGG) site of plasmid pUC19. HindIII-EcoRI restriction fragments containing these inserts have been used as substrates for footprinting experiments using DNase I, DNase II, and micrococcal nuclease as probes. These present good mithramycin binding sites (GGG) flanking repetitive regions to which the drug does not bind. In each case, mithramycin footprints are observed at the CCC/GGG sites, which are not affected by the nature of the surrounding sequences. Some weaker binding is detected at TCGA and ACCA sites and at regions of alternating GA. No binding is found to regions of alternating GT. An.Tn inserts (n = 23 or 69) are normally resistant to cleavage by all these probes; in the presence of mithramycin, a dramatic increase in DNase I cleavage is observed throughout the entire insert and is indicative of an alteration in DNA structure. Similar changes are seen with DNase II and micrococcal nuclease. These changes cannot be explained by invoking changes in the ratio of free substrate to cleavage agent. In contrast, cleavage of (GA)n.(CT)n and (GT)n.(AC)n inserts is not affected by drug binding. The results are consistent with a model in which mithramycin causes dramatic changes in the width of the DNA minor groove, generating a structure which has some properties of A-DNA, and suggest that this can be propagated into surrounding DNA regions in a sequence-dependent manner. The structural alterations with An.Tn are highly cooperative and can be transmitted over at least three turns of the DNA helix.

Base Composition↗
PubMed1991

The effects of actinomycin on the structure of dAn.dTn and (dA-dT)n regions surrounding its GC binding site. A footprinting study.

The effect of actinomycin on the structure of DNA fragments containing the sequences (AT)5GC(AT)5, (TA)5GC(TA)5, A9GCT9, and T9GCA9, cloned into the SmaI site of pUC19, has been studied by footprinting analysis using a variety of probes known to be sensitive to DNA structure. In each case clear footprints are found around the central GC sites. DNase I cleavage of fragments containing alternating AT shows much greater cutting at ApT than TpA; in the presence of actinomycin, although this preference is retained, there is a large increase in the cutting efficiency at the closest TpA steps. DNase I cleavage in homopolymeric regions of A and T, which is normally very poor, is greatly enhanced by drug binding. With T9GCA9 the enhancements are propagated in both directions, whereas changes are only found to the 5'-side of the GC site in A9GCT9. The results are confirmed by similar experiments with micrococcal nuclease and DNase II. Small increases in sensitivity to diethylpyrocarbonate are found at adenines proximal to GC. Experiments performed at 4 degrees C suggest that conformational changes are a necessary consequence of drug binding.

Base Sequence↗
PubMed1990

Effects of sequence selective drugs on the gel mobility of a bent DNA fragment.

The effects of various drugs on the structure of a bent DNA fragment have been investigated by studying DNA mobility in polyacrylamide gels. This DNA fragment has an anomalously slow rate of migration on account of its phased runs of adenines. Nogalamycin and echinomycin increase the gel mobility of kinetoplast DNA suggesting that the bending has been removed. Mithramycin, actinomycin, distamycin and ethidium have either no effect or cause a further reduction in mobility. These results are compared with other, non-bent DNA species which always show a decrease in gel mobility in the presence of DNA binding drugs.

Adenine↗
PubMed1990

Long (dA)n.(dT)n tracts can form intramolecular triplexes under superhelical stress.

Plasmids containing long tracts of (dA)n.(dT)n have been prepared and their conformations examined in linear and supercoiled DNA using a series of chemical and enzymic probes which are known to be sensitive to unusual DNA structures. Under superhelical stress and in the presence of magnesium the sequence T69.A69 adopts a conformation at pH 8.0 consistent with the formation of an intramolecular DNA triplex. Site specific cleavage of the supercoiled plasmid by single-strand specific nucleases occurs within the A.T insert; the 5'-end of the purine strand is sensitive to reaction with diethylpyrocarbonate while the central 5-6 bases of the pyrimidine strand are reactive to osmium tetroxide. By contrast shorter inserts of A33.T33 and A23.T23 do not appear to form unusual structures.

Base Sequence↗
PubMed1990

Clinical and pathologic features of primary gastric rhabdomyosarcoma.

The authors report a case of a collision tumor composed of a primary gastric rhabdomyosarcoma intermingled with an adjacent infiltrating gastric adenocarcinoma. Only eight cases of gastric rhabdomyosarcoma have been reported previously and little information is recorded about the behavior of this tumor. Gastric rhabdomyosarcoma has several distinctive clinical features. It is a highly aggressive tumor (median survival, 2.5 months) occurring in both children and adults and frequently presents as metastatic disease to lung or cervical lymph node. The initial diagnosis is often difficult to establish, especially on biopsy material. In several instances, the correct diagnosis was established only at autopsy. The authors' patient was correctly diagnosed during life and received considerable benefit from cyclophosphamide, doxorubicin, and vincristine (CAV) therapy and survived for 26 months after diagnosis. Two of the previously reported eight cases of primary gastric rhabdomyosarcoma and this case have adjacent admixed glandular gastric adenocarcinomas. Since three of the nine known cases of primary gastric rhabdomyosarcoma have an adjacent admixed gastric adenocarcinoma, there appears to be a greater than chance association between these two tumors.

Adenocarcinoma↗
PubMed1990

Interaction of berenil with the tyrT DNA sequence studied by footprinting and molecular modelling. Implications for the design of sequence-specific DNA recognition agents.

We have developed a technique of partially-restrained molecular mechanics enthalpy minimisation which enables the sequence-dependence of the DNA binding of a non-intercalating ligand to be studied for arbitrary sequences of considerable length (greater than = 60 base-pairs). The technique has been applied to analyse the binding of berenil to the minor groove of a 60 base-pair sequence derived from the tyrT promoter; the results are compared with those obtained by DNAse I and hydroxyl radical footprinting on the same sequence. The calculated and experimentally observed patterns of binding are in good agreement. Analysis of the modelling data highlights the importance of DNA flexibility in ligand binding. Further, the electrostatic component of the interaction tends to favour binding to AT-rich regions, whilst the van der Waals interaction energy term favours GC-rich ones. The results also suggest that an important contribution to the observed preference for binding in AT-rich regions arises from lower DNA perturbation energies and is not accompanied by reduced DNA structural perturbations in such sequences. It is therefore concluded that those modes of DNA distortion favourable to binding are probably more flexible in AT-rich regions. The structure of the modelled DNA sequence has also been analysed in terms of helical parameters. For the DNA energy-minimised in the absence of berenil, certain helical parameters show marked sequence-dependence. For example, purine-pyrimidine (R-Y) base pairs show a consistent positive buckle whereas this feature is consistently negative for Y-R pairs. Further, CG steps show lower than average values of slide while GC steps show lower than average values of rise. Similar analysis of the modelling data from the calculations including berenil highlights the importance of DNA flexibility in ligand binding. We observe that the binding of berenil induces characteristic responses in different helical parameters for the base-pairs around the binding site. For example, buckle and tilt tend to become more negative to the 5'-side of the binding site and more positive to the 3'-side, while the base steps at either side of the centre of the site show increased twist and decreased roll.

Amidines↗