Oligonucleotide Synthesis

Volume 288 of the series Methods in Molecular Biology pp 319-342

DNase I Footprinting of Small Molecule Binding Sites on DNA

  • Christian BaillyAffiliated withINSERM U-524, Institut de Recherches sur le Cancer de Lille
  • , Jérôme KluzaAffiliated withINSERM U-524, Génétique Moléculaire et Approches Thérapeutiques de éopathies Malignes
  • , Christopher MartinAffiliated withDepartment of Pharmacology, University of Cambridge
  • , Thomas EllisAffiliated withDepartment of Pharmacology, University of Cambridge
  • , Michael J. WaringAffiliated withDepartment of Pharmacology, University of Cambridge

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Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.

Key Words

Nuclease DNase I footprinting DNA-binding drugs DNase I DNase II micrococcal nuclease anticancer antibiotic actinomycin D endonuclease electrophoresis polyacrylamide gels echinomycin cooperativity drug-DNA recognition reverse transcriptase DNA polymerase I diaminopurine, inosine electroelution dimethylsulfate densitometric analysis capillary electrophoresis sequence selectivity DNA recognition