Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.

Abstract

A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly endlabeled restriction fragments and partial digestion of the drug fragment complex with DNase I. Microdensitometry of autoradiograms produced after electrophoretic separation of digestion products allows determination of sequences that are affected by drug binding. The feasibility of the technique for locating small ligands bound to DNA and its eventual use as a quantitative thermodynamic approach to studying ligand binding to heterogeneous DNA as a function of sequence is illustrated by using actinomycin D and Netropsin.The antitumor agent actinomycin D, used in the treatment of Wilms tumor (1), and the bactericidal/antiviral (2) compound Netropsin are thought to function, in vivo, by binding, in an equilibrium fashion, to cellular DNA. It is of fundamental significance to determine the extent to which such compounds have well-defined nucleotide base sequence specificities on natural heterogeneous-sequence DNA. These agents leave no record of their residence positions on the helix-i.e., they neither cleave the DNA backbone as does bleomycin (3, 4) nor covalently modify the bases as does mitomycin (5). Therefore an accurate determination of the locations of preferential interaction sites has been difficult, relying largely on homopolymer studies.Another approach to the study of the sequence specificity of drug DNA equilibrium interactions is presented in this paper. The DNase protection technique developed by Galas and Schmitz (6, 7) for determining the binding sites of proteins on a DNA helix has been applied to the problem of locating small equilibrium binders such as actinomycin D and Netropsin. The method derives from the strategy of Maxam and Gilbert (8) for rapid DNA sequence analysis. A specific 142-base-pair (bp) restriction fragment is used as template, and the endonuclease DNase I is the binding site probe. Enzyme digestion conditions are rigorously controlled in order to approximate "single-hit" kinetic criteria and to facilitate quantitative evaluation of the extent of nuclease activity at a site containing the drug. The procedure is further developed by the application of microdensitometric scanning of gel autoradiograms to help visualize band intensity changes and to begin to determine thermodynamic parameters for small ligand binding as a function of base sequence.MATERIALS AND METHODS Materials. Calf thymus DNA and DNase I were purchased from Sigma. Actinomycin D and Netropsin were obtained from the National Cancer Institute and J. A. Bush (Bristol-Myers, Syracuse, NY), respectively. Both drugs were used without further purification. Restriction enzymes and phage T4 polynucleotide kinase were purchased from Bethesda Research Laboratories and used in accordance with supplier specifications.[y-32P]ATP having a specific activity of ==2,500 Ci/mmol (1 Ci = 3.7 X 101' Bq) was obtained from New England Nuclear. The concentrat...

Distamycin-induced inhibition of homeodomain-DNA complexes.

“…Netropsin is a close relative of distamycin containing two methylpyrrolecarboxamide rings and a guanidinium group in place of the third methylpyrrole moiety of distamycin (Zimmer, 1975) which exhibits very similar specificities for AT-rich regions of double helical DNA to distamycin (Van Dyke et al, 1982;Lane et al, 1983). In order to check whether distamycin and netropsin inhibit the specific Antp HD-DNA binding equally well we performed the same competition experiments with netropsin.…”

Selective inhibition of topoisomerases from Pneumocystis carinii compared with that of topoisomerases from mammalian cells

“…While intercalators cause major structural distortions, ligands which interact with DNA by groove binding effect only minor changes such as widening of the minor groove of DNA and localized bending of the DNA helix (29). Despite the minimal conformational changes caused by these agents, both distamycin and netropsin have been shown to alter DNA-directed enzyme reactions such as DNase I cleavage (15,24) and RNA polymerase transcription (8,37) In terms of DNA binding, previous studies have determined that these compounds bind in the minor groove of B-DNA with an AT base pair preference and do not intercalate (14). It has been suggested that the size of the DNA-binding site by the minor groove-binding compound is important to their potencies as antimicrobial agents (42).…”

Comparison of binding sites in DNA for berenil, netropsin and distamycin

“…We are at a loss to provide any cogent explanation and respectfully note the seeming complexity of the recognition processes involved. For the time being it must suffice to reiterate the belief [7,13,20] that apparent site sizes, and effects on flanking nucleotides, not only reflect the extent of true specific binding sites but are also governed by steric disturbances to the DNA helix which influence its ability to accommodate nucleases 1191 and serve as a substrate for cutting.…”

“…It is generally reckoned that berenil should bind preferentially to A+T-rich DNA [l, 5, 111. In this paper, we present the first (to our knowledge) footprinting study on the binding of berenil to DNA. In addition, because some previous footprinting work using netropsin and distamycin indicated that the binding-site sizes are longer than just the A + T-rich zones [7,81 we have investigated the acceptability of G . C base pairs in the preferred binding sites for all these drugs, whether within or adjacent to the A + T-rich zones, as well as the extent of conformational changes resulting from ligand binding.…”