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Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation

Academic Article
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Overview

authors

  • Pei, D. H.
  • Corey, D. R.
  • Schultz, Peter

publication date

  • 1990

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

abstract

  • A Lys-84----Cys mutant staphylococcal nuclease was selectively linked to the 5' and/or 3' terminus of a thiol-containing polypyrimidine oligonucleotide via a disulfide bond. The oligonucleotide-staphylococcal nuclease adduct is capable of binding to a homopurine-homopyrimidine region of Watson-Crick duplex DNA by the formation of a triple-helical structure. Upon the addition of Ca2+, the nuclease cleaves DNA at sites adjacent to the homopurine tract. Specific double-strand cleavage occurred predominantly at A + T-rich sites to the 5' side of the homopurine tract for both the 5'-derivatized and the 5',3'-diderivatized nucleases; the 3'-derivatized nuclease gave no cleavage. The cleavage pattern is asymmetric and consists of multiple cleavage sites shifted to the 5' side on each strand, centered at the terminal base pair of the binding site. Microgram amounts of plasmid pDP20 DNA (4433 base pairs) containing a homopurine-homopyrimidine tract were selectively cleaved by a semisynthetic nuclease with greater than 75% efficiency at room temperature within 1 hr. Cleavage reaction conditions were optimized with respect to pH, temperature, reaction times, and reaction components. Semisynthetic nucleases of this type should provide a powerful tool in chromosomal DNA manipulations.

subject areas

  • Base Sequence
  • Cysteine
  • DNA
  • Kinetics
  • Lysine
  • Micrococcal Nuclease
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nucleic Acid Conformation
  • Nucleic Acid Denaturation
  • Oligodeoxyribonucleotides
  • Oligonucleotide Probes
  • Substrate Specificity
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Identity

PubMed Central ID

  • PMC55273

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.87.24.9858

PubMed ID

  • 2124705
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Additional Document Info

start page

  • 9858

end page

  • 9862

volume

  • 87

issue

  • 24

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