Drosophila engrailed-1,10-phenanthroline chimeras as probes of homeodomain-DNA complexes.
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.
Pan
C Q
CQ
Molecular Biology Institute, University of California at Los Angeles 90095-1570, USA.
Landgraf
R
R
Sigman
D S
DS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007301:Insect Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010618:Phenanthrolines / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D007301:Insect Hormones; D010618:Phenanthrolines; D014157:Transcription Factors; D004247:DNA; D003545:Cysteine; C025205:1,10-phenanthroline