Drosophila engrailed‐1, 10‐phenanthroline chimeras as probes of homeodomain‐DNA complexes

Clark Q. Pan, Ralf Landgraf, David S. Sigman

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

We have converted the Drosophila engrailed homeodomain into a sequence specific nuclease by linking the protein to the chemical nuclease 1,10- phenanthroline-copper (OP-Cu). Unique cysteines were introduced at six positions into the homeodomain by site directed mutagenesis for the covalent attachment of OP-Cu. The varied DNA-binding affinity and specificity of these mutants and the DNA cleavage pattern of their OP-Cu derivatives allowed us to assess the crystal structure of the engrailed homeodomain-DNA complex. We have also achieved site-specific double-stranded DNA scission with one of the homeodomain mutants, E28C, which has the potential of being used to identify engrailed binding sites in the genome. Because the homeodomain is so well conserved among members of the homeodomain-containing protein family, other homeodomain proteins can be converted into nucleases by attaching OP-Cu at position 28 of their homeodomains.

Original languageEnglish (US)
Pages (from-to)2279-2288
Number of pages10
JournalProtein Science
Volume4
Issue number11
DOIs
StatePublished - Nov 1995
Externally publishedYes

Keywords

  • 1,10-phenanthroline-copper
  • chemical nuclease
  • homeodomain
  • protein-DNA interaction

ASJC Scopus subject areas

  • Biochemistry

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