A 96-well DNase I footprinting screen for drug-DNA interactions

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive 'one compound-one line' scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.     

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.     

A recommended workflow for DNase I footprinting using a capillary electrophoresis genetic analyzer

Fragment analysis was developed to determine the sizes of DNA fragments relative to size standards of known lengths using a capillary electrophoresis genetic analyzer. This approach has since been adapted for use in DNA footprinting. However, DNA footprinting requires accurate determination of both fragment length and intensity, imposing specific demands on the experimental design. Here we delineate essential considerations involved in optimizing the fragment analysis workflow for use in DNase I footprinting to ensure that changes in DNase I cleavage patterns may be reliably identified.     

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.     

'Footprinting' proteins on DNA with peroxonitrous acid.

The peroxonitrite anion (ONOO-) is a stable species in alkaline solution that quickly generates a strong oxidant at neutral pH. A convenient procedure for the preparation of ONOOK has been developed based on the procedure of Keith & Powell [(1969) J. Chem. Soc. A, 90], which when added to a sample of duplex DNA buffered at neutral pH rapidly generates a strong oxidant capable of nonspecifically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the cl-repressor (cl) of phage lambda with the right operator, OR. In addition, we show that the individual-site binding isotherms determined by quantitative DNase I, Fe-EDTA and ONOOK footprinting are identical within experimental error. The identical isotherms obtained with the three different reagents with greatly differing sampling times indicates that the sampling time of the footprinting probe need not be short relative to the kinetic dissociation constants that govern protein-DNA interactions.     

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.     

Sequence selective binding of ditrisarubicin B to DNA: comparison with daunomycin

DNase I footprinting has been used to examine the sequence selective binding of ditrisarubicin B, novel anthracycline antibiotic, to DNA. At 37°C no footprinting pattern is observed, the drug protects all sites from enzymic cleavage with equal efficiency. At 4°C a footprinting pattern is induced with low drug concentrations which is different from that produced by daunomycin. The best binding sites contain the dinucleotide step GpT (ApC) and are located in regions of alternating purines and pyrimidines.     

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.     

Structural changes and enhancements in DNase I footprinting experiments.

In footprinting experiments, an increase in DNA cleavage with addition of ligand to a system may be due to a ligand-induced structural change. Ligand binding also enhances cleavage by displacing the cleavage agent from ligand-binding sites, thus increasing its concentration elsewhere. The theory and characteristics of this mass-action enhancement are given, and it is shown how it may be recognized. Results of DNase I footprinting of small oligomers, with actinomycin D as ligand, are analyzed to reveal which enhancements are due to mass action, and which can reasonably be ascribed to structural changes. Patterns in the footprinting plots from our experiments on actinomycin D binding to a 139-base-pair DNA fragment (with DNase I as a probe) are studied in the same way. The likely origins of these patterns are discussed, as are enhancements occurring with other probes commonly used in footprinting experiments.     

Rapid measurement of deoxyribonuclease I activity with the use of microchip electrophoresis based on DNA degradation

Deoxyribonuclease I (DNase I) activity in serum has been shown to be a novel diagnostic marker for the early detection of acute myocardial infarction (AMI). However, the conventional method to measure DNase I activity is time-consuming. In the current study, to develop a rapid assay method for DNase I activity for clinical purposes, a microchip electrophoresis device was used to measure DNase I activity. Because DNase I is an endonuclease that degrades double-stranded DNA endo-nucleolytically to produce oligonucleotides, degradation of the DNA standard caused by DNase I action was detected using microchip electrophoresis. We detected DNase I activity within 10 min. This is the first study to apply microchip electrophoresis for the detection of DNase I activity; furthermore, it seems plausible that reduction of analysis time for DNase I activity could make this novel assay method using microchip electrophoresis applicable in clinical use.     

Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5-thyminyl-5,6-dihydrothymine) but not cyclobutane pyrimidine dimers in UV-irradiated DNA

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py lozengePy) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py lozengePy-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the (32)P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.     

DNA footprint enhancement using tandem binding sites.

A concatenated DNA fragment containing a five-repeat binding site was used for DNase I footprinting. Under the same conditions, the tandem repeat assay greatly enhanced the DNA footprint as compared with a native DNA sequence with only one binding site. This technique provides an approach for improving poor DNA footprints.