Sequence Specificity of Actinomycin D and Netropsin Binding to pBR322 DNA Analyzed by Protection from DNase I

A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly end-labeled restriction fragments and partial digestion of the drug fragment complex with DNase I. Microdensitometry of autoradiog...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1983-06, Vol.80 (11), p.3260-3264
Main Authors: Lane, Michael J., Dabrowiak, James C., Vournakis, John N.
Format: Article
Language:English
Subjects:
Actinomycin
Animals
Autoradiography
Base Sequence
Binding sites
Biochemistry
Cattle
Chemical Phenomena
Chemistry
Dactinomycin
Deoxyribonuclease I
DNA
Endodeoxyribonucleases - metabolism
Enzymes
Gastrointestinal agents
Gels
Guanidines
Ligands
Molecules
Netropsin
Optical density
Phosphorus Radioisotopes
Plasmids
Substrate Specificity
Thymus Gland
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Description
Summary:A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly end-labeled 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.80.11.3260