Rate enhancements in the DNase I footprinting experiment.

Footprinting experiments for DNase I digests of a 139-base-pair segment of pBR-322 DNA in the presence of either netropsin or actinomycin D were carried out. Plots of oligonucleotide concentration as a function of drug concentration were analyzed to study the enhancement in cleavage rates at approximately 30 sites, accompanying drug binding at other sites. The pattern of enhancements is not consistent with drug-induced DNA structural changes, but agrees with a redistribution mechanism involving DNase I. Since the total number of enzyme molecules per fragment remains unchanged, drug binding at some sites increases the enzyme concentration at other sites, giving rise to increased cleavage. The consequences of the redistribution mechanism for analysis of footprinting experiments are indicated.     

Actinomycin D induced DNase I cleavage enhancement caused by sequence specific propagation of an altered DNA structure.

Two DNA hexadecamers containing one central 5'-GC-3' base step have been examined by footprinting methodology in the presence and absence of actinomycin D. The results of these studies, coupled with imino proton NMR measurements indicate that the antitumor drug causes a change in DNA conformation at a distance from the actinomycin intercalation site in a molecule of sequence d[ATATATAGCTATATAT] that does not occur in d[AAAAAAAGCTTTTTTT]. The experiments demonstrate that DNase I rate enhancements associated with actinomycin D binding are caused by ligand alteration of equilibrium DNA structure.     

Quantitative footprinting analysis. Binding to a single site

The theory for measuring ligand binding constants from footprinting autoradiographic data associated with a single binding site is derived. If the ligand and DNA cleavage agent compete for a common site, the spot intensities are not proportional to the amount of DNA not blocked by ligand. The analysis of a single site is experimentally illustrated by using results for the anticancer drug actinomycin D interacting with the duplex d(TAGCGCTA)2 as probed with the hydrolytic enzyme DNase I.     

Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA.

DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA.     

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.     

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.     

Properties of DNase I digestion of the deoxyoligonucleotide: 5''d(ATCGTACGAT)2(3'').

Deoxyribonuclease I digestion of the deoxyoligodecamer 5'd(ATCGTACGAT)2(3') has been examined in detail to study the kinetic and structural properties of this enzyme substrate system in solution. In addition, these studies have defined, in general, those DNase I conditions to be used in future drug-DNA footprinting experiments. Special attention has been taken of those properties of DNase I that are critical for quantitation of ligand binding to small DNA fragments, and that aid in designing oligomers to be used in footprinting experiments. Enzyme activity was observed at all phosphodiester bonds in the decamer studied with varying affinity, except for the first four bonds at the 5' end of the oligomer. The DNA substrate concentration is always in excess, in order to achieve conditions of no more than one DNase I cleavage per DNA molecule. Reactions were controlled so that 65% or more of the initial amount of decamer substrate remained after DNase I digestion. It was observed that the rate of enzyme reactivity decreases with digestion time and is sensitive to the experimental conditions.     

Footprinting: a method for determining the sequence selectivity, affinity and kinetics of DNA-binding ligands

Footprinting is a simple method for assessing the sequence selectivity of DNA-binding ligands. The method is based on the ability of the ligand to protect DNA from cleavage at its binding site. This review describes the use of DNase I and hydroxyl radicals, the most commonly used footprinting probes, in footprinting experiments. The success of a footprinting experiment depends on using an appropriate DNA substrate and we describe how these can best be chosen or designed. Although footprinting was originally developed for assessing a ligand's sequence selectivity, it can also be employed to estimate the binding strength (quantitative footprinting) and to assess the association and dissociation rate constants for slow binding reactions.     

DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected. Distamycin recognizes four major regions rich in A + T residues. Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites. These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin. The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.     

Site-specific binding constants for actinomycin D on DNA determined from footprinting studies.

We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at approximately 7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3' end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3' end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about approximately 120 base pairs of the restriction fragment (approximately 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5'----3') GC, with TGCT having the highest binding constant, 6.4 x 10(6) M-1. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.     

Comparison of binding sites in DNA for berenil, netropsin and distamycin. A footprinting study

Techniques of DNase I and micrococcal nuclease footprinting have been used to compare the binding sites for berenil, netropsin and distamycin on two different DNA fragments. Each ligand binds to the A + T-rich zones which contain clusters of at least four A.T base pairs. Neither guanosine nor cytidine nucleotides appear to be allowed within the A + T-rich runs which constitute the preferred binding sites, although they are sometimes protected from DNase I cleavage in neighbouring regions. Berenil and netropsin share with distamycin the property of causing enhanced rates of cleavage at certain sequences flanking their binding sites. There are significant differences in the concentrations of each ligand required to produce defined patterns of protection, seemingly dependent upon the nature (and possibly the gross base composition) of the piece of DNA being used in the experiment.     

Quantitative footprinting analysis of the netropsin-DNA interaction

The results of a series of quantitative footprinting experiments of the netropsin-DNA interaction as studied using two different DNA cleaving probes, the enzyme DNase I and a cationic manganese porphyrin complex, are described. Plots of the relative change in oligonucleotide concentration as a function of drug concentration, covering approximately 110 base pairs of a DNA restriction fragment, revealed netropsin induced changes in the cleavage rates of both probes. These appeared as inhibitions for the binding sites, enhancements where no binding took place, and enhancement/inhibitions for the weak binding sites. Determination of the concentration of drug necessary to reduce the amount of a particular oligomer to half of its initial value allowed a ranking of the affinities of the various binding sites on the fragment. In addition to uncovering the location of a number of overlapping netropsin binding sites, the data allowed additional insight on the manner in which both probes alter their DNA cleavage rates in the drug-footprinting experiment.     

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.     

Actinomycin D induced DNase I hypersensitivity and asymmetric structure transmission in a DNA hexadecamer.

DNase I cleavage rates and nmr chemical shifts are shown to change for DNA sequences distal to an intercalated actinomycin D molecule in a duplex hexadecamer upon drug binding. Both sets of observations suggest that the source of these changes is a DNA-mediated structural response. The nmr results imply the response is transmitted preferentially in a 5'-to-3' direction from the drug binding site. An inequivalent response of the two strands to a ligand-induced conformational change immediately suggests a mechanism for distinguishing the sense and antisense strands of DNA.     

Footprinting protein-DNA complexes using the hydroxyl radical

Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA-protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.     

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.     

Footprinting reveals that nogalamycin and actinomycin shuffle between DNA binding sites.

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.     

Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting.

We have investigated the kinetics of dissociation of actinomycin D from DNA by a variation of the footprinting technique. Complexes of actinomycin with a radiolabelled DNA fragment (tyrT) were dissociated by addition of a large excess of unlabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37 degrees C, 20 degrees C and 4 degrees C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20 degrees C. In contrast, the dissociation of distamycin was too fast to measure (< 5 s) even at 4 degrees C.