Biomedical subjects
Publications and source records attributed to K R Fox.
DNAase I footprinting of restriction enzymes has been achieved by using calcium containing digestion buffers so that the enzymes bind to but do not cleave DNA. EcoR1 produces a footprint 17 bases long, overestimating the region of contact with DNA by about 7-8 base pairs. Restriction enzymes HaeIII and HinP1 generate smaller footprints of 15 and 13 base pairs respectively.
The reactivity of the 160 bp tyrT DNA fragment towards diethyl pyrocarbonate (DEPC) has been investigated in the presence of bis-intercalating quinoxaline antibiotics and the synthetic depsipeptide TANDEM. At moderate concentrations of each ligand, specific purine residues (mainly adenosines) exhibit enhanced reactivity towards the probe, and several sites of enhancement appear to be related to the sequence selectivity of drug binding. Further experiments were performed with echinomycin at pH 5.5 and 4.6 to facilitate the protonation of cytosine required for formation of Hoogsteen GC base pairs. No significant increase in reactivity was observed under these conditions. Additionally, no protection of deoxyguanosine residues from methylation by dimethyl sulphate was observed in the presence of echinomycin. We conclude that the structural anomaly giving rise to drug-dependent enhanced DEPC reaction is not simply the formation of Hoogsteen base pairs adjacent to antibiotic binding sites. Nor is it due to a general unwinding of the double helix, since we show that conditions which are supposed to unwind the helix lead to a uniform increase in purine reactivity, regardless of the surrounding nucleotide sequence.
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DNA structural changes induced by bleomycin have been investigated using diethylpyrocarbonate and permanganate as probes under conditions in which the antibiotic binds to, but does not cut the DNA. Diethyl-pyrocarbonate shows an enhanced reaction with adenines in the presence of the antibiotic in the sequences GTA greater than GCA greater than GAA, on the 3' side of the drug cutting site (GPy). Permanganate ions display an enhanced reactivity at the second pyrimidine of the sequence GPyPy. The results are consistent with a model in which bleomycin distorts the structure of the base pair on the 3' side of its binding site.
We have examined the binding of luzopeptin, an antitumor antibiotic, to five DNA fragments of varying base composition. The drug forms a tight, possibly covalent, complex with the DNA causing a reduction in mobility on nondenaturing polyacrylamide gels and some smearing of the bands consistent with intramolecular cross-linking of DNA duplexes. DNAase I and micrococcal nuclease footprinting experiments suggest that the drug binds best to regions containing alternating A and T residues, although no consensus di- or trinucleotide sequence emerges. Binding to other sites is not excluded and at moderate ligand concentrations the DNA is almost totally protected from enzyme attack. Ligand-induced enhancement of DNAase I cleavage is observed at both AT and GC-rich regions. The sequence selectivity and characteristics of luzopeptin binding are quite different from those of echinomycin, a bifunctional intercalator of related structure.
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DNase I footprinting studies employing several DNA fragments have confirmed that nogalamycin binds preferentially to regions of DNA containing alternating purines and pyrimidines. Arugomycin and viriplanin A, related compounds which contain additional sugar residues at both ends of the molecule, produce similar patterns of nuclease protection though at higher drug concentrations. The pattern induced by decilorubicin, which has charged groups at both ends of the aglycone, differs in many details and this analogue appears to display a modified DNA sequence selectivity. The results have been confirmed by similar studies using DNase II. All four compounds increase the susceptibility of certain adenine residues to modification by diethylpyrocarbonate. The results suggest an intercalative mode of binding for these nogalamycin analogues, and reveals an increased complexity in compounds which can bind to DNA by this mechanism.
Controversy exists over the effect of definitive radiotherapy on the ability to administer full doses of adjuvant chemotherapy in primary breast cancer. Ninety-six consecutive women with clinical stage I and II breast cancer were treated with radiotherapy plus chemotherapy. Three combinations of drugs were used: cyclophosphamide and 5-fluorouracil (CF); cyclophosphamide, methotrexate, and 5-fluorouracil (CMF); or cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone (CMFP). Chemotherapy consisted of two cycles of CF (cyclophosphamide at a dosage of 100 mg/m2 orally on days 1-14+5-fluorouracil at 600 mg/m2 iv on days 1 and 8) during concurrent radiotherapy, followed by six cycles of CMFP (same CF dosages+methotrexate at 40 mg/m2 iv on days 1 and 8+prednisone at 40 mg/m2 orally on days 1-14). The study included 63 premenopausal and 33 postmenopausal patients; 72 had 1-3 positive nodes, had greater than or equal to 4 positive nodes, and 9 had negative nodes and negative estrogen receptors. The mean CF doses delivered during concurrent radiotherapy were 95% of the optimal doses, and the mean CMF doses administered during the six cycles after radiotherapy were 89%. The CMF was delivered at level I (greater than or equal to 85% of optimal doses) to 73% of the patients. With a median follow-up of 36 months, 16 relapses have been observed. Two of these patients had treatment failure only in the breast or axilla and are disease free after mastectomy. Of the 72 patients with 1-3 positive nodes, 10 relapsed in distant sites, while 4 of 15 patients with greater than or equal to 4 positive nodes have had distant failure.(ABSTRACT TRUNCATED AT 250 WORDS)
The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.
The cutting pattern produced by micrococcal nuclease on three DNA fragments has been determined in the absence and presence of various DNA-binding drugs. The enzyme itself cuts almost exclusively at pA and pT bonds, showing a greater activity at (A-T)n than in homopolymeric runs of A and T. Each drug produces distinct changes in the cleavage pattern. The protected regions can not be pinpointed with sufficient precision to assess the exact drug-binding sites on account of the sequence selectivity of the enzyme, although where a direct comparison is possible these include most of those seen as DNAase I footprints. The enzyme is most useful for assessing the selectivity of drugs which bind to AT-rich regions. Several drugs protect the DNA from micrococcal nuclease attack in regions which do not contain their acknowledged best binding sites. It appears that micrococcal nuclease is sensitive to the existence of secondary drug-binding sites which are not evident with other footprinting techniques.
The binding of phleomycin and bleomycin to DNA has been investigated by studying their effects on cleavage by DNAase I and micrococcal nuclease. In the presence of cobalt, cleavage of DNA by the antibiotics is suppressed, yet they still provide protection from nuclease attack in regions surrounding the drug cleavage sites. We conclude that cleavage by phleomycin occurs at bonds around which the antibiotic is already selectively bound.
The DNA sequence preferences of the compound meso-tetra-(4-N-methyl(pyridyl) porphyrin and its nickel complex have been investigated by means of footprinting experiments on several DNA fragments, using DNAase I and micrococcal nuclease as footprinting agents. A complex pattern of both AT and GC-protected sites was found. Ligand-induced long-range conformational changes were inferred in several instances to be related to the observed large-scale blockages of enzymatic cutting.
Footprinting experiments employing DNAase I have been performed at 4 degrees C. At this temperature several simple intercalating ligands, including both ethidium and proflavine, can be seen to induce marked changes in the pattern of cleavage. From an analysis of the changes in patterns of digestion by DNAase I we deduce that ethidium binds best to regions of mixed nucleotide sequence, especially those containing alternating purines and pyrimidines. Binding seems to be weakest to poly dA sequences which consequently appear as regions of relatively enhanced cleavage. Attempts to reproduce these changes using DNAase II as a footprinting tool were unsuccessful.
Five broth media and two solid media were compared for their ability to quantitatively recover total coliform bacteria from chemically treated water. M-Endo LES and mT7 media were used in the membrane filter technique. Lauryl tryptose broth, lactose broth, presence-absence broth, lactose broth with twice the amount of lactose, and lauryl tryptose broth with twice the amount of sodium lauryl sulfate were used in the fermentation tube procedure. The differences in recovery were not significant for the five broth media and M-Endo LES agar. The M-Endo LES and mT7 media were not significantly different; however, the five broth media did yield significantly higher counts than mT7.
Four DNA restriction fragments, designated tyrT, pTyr2, pUC13, and Xbs1, have been used as substrates for footprinting studies with DNase I in the presence of the anthracycline antibiotic nogalamycin. With each fragment a distinct pattern of antibiotic-protected binding sites is observed, but no concensus sequence emerges from the data. All sites are located in regions of alternating purine-pyrimidine sequence, most commonly associated with the dinucleotide steps TpG (CpA) and GpT (ApC), suggesting that the preferred binding sites may contain all four nucleotides and/or that peculiarities of the dynamics of DNA conformation at alternating sequences may be critical for nogalamycin binding. Some concentration dependence of footprinting patterns is evident, in contrast to previous studies with a variety of sequence-specific ligands. Enhanced susceptibility to attack by DNase I is commonly observed at sequences flanking strong antibiotic-binding sites. Nogalamycin selectively inhibits cleavage of DNA at certain guanine-containing sequences by the G-specific photosensitized reaction with methylene blue. Comparison of these effects with its action on the G-specific reaction with dimethyl sulfate suggests that the amino sugar moiety of nogalamycin may be preferentially located in the minor helical groove at some binding sites but in the major groove at others.
The interaction has been studied of several anthraquinone-based intercalating drugs, including the anti-cancer agent mitoxantrone, with defined sites of DNA. A 160 base pair DNA sequence from tyrT was employed for footprinting with DNase I. The anthraquinones had aminoalkylamino substituents in various positions of the ring system. Inhibition of enzymatic cutting of the DNA was observed at various positions on the sequence, mostly around some of the pyrimidine-3',5'-purine sites. Enhancements to cutting were observed clustered around AT-rich regions. The compounds showed differences in detailed footprinting behaviour, which have been related to differences in their mode of interaction with DNA as found in earlier computer modelling studies.
The hypothesis that sequence-selective DNA-binding antibiotics locate their preferred binding sites by a process involving migration from nonspecific sites has been tested by footprinting with DNAase I. Footprinting patterns on the tyrT DNA fragment produced by nogalamycin and actinomycin change with time after mixing the antibiotic with the DNA. Sites of protection as well as enhanced cleavage are seen to develop in a fashion which is both temperature and concentration-dependent. At certain sites cutting is transiently enhanced, then blocked. Limited evidence for slow reaction with echinomycin and mithramycin is presented, but the kinetics of footprinting with daunomycin and distamycin appear instantaneous. The feasibility of adducing direct evidence for shuffling by footprinting seems to be governed by slow dissociation of the antibiotic-DNA complex. It may also be dependent upon the mode of binding, be it intercalative or non-intercalative in character.
Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N-MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA.