Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent

Proc Natl Acad Sci U S A. 1990 Apr;87(8):2882-6. doi: 10.1073/pnas.87.8.2882.

Abstract

Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Coliphages / metabolism
  • Cyclic AMP Receptor Protein / metabolism*
  • Cysteine
  • DNA, Viral / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Phenanthrolines
  • Protein Conformation
  • Restriction Mapping

Substances

  • Cyclic AMP Receptor Protein
  • DNA, Viral
  • DNA-Binding Proteins
  • Phenanthrolines
  • Cysteine
  • 1,10-phenanthroline