Effects of length and position of an extended linker on sequence-selective DNA recognition of zinc finger peptides.
Engineered zinc finger proteins revealed that a linker sequence connecting zinc finger units has a significant effect on the DNA binding property of the protein. The recognition for a noncontiguous DNA target beyond the current recognition code of zinc finger proteins has never been determined because of the limitation of a zinc finger framework. DNA recognition of zinc finger proteins is limited only to a contiguous subset of three base pairs. We propose the recognition for a noncontiguous DNA target by inserting amino acids into the canonical linker between zinc finger units. The sequence selectivity of the new zinc finger peptides was evaluated by gel mobility shift assays. DNase I footprinting analyses clearly showed different DNA binding of various linker-extended zinc finger peptides. The application of a SPR measurement also revealed a DNA sequence selectivity of peptides. Insertion of three amino acids is enough for recognition of a noncontiguous DNA target with sequence selectivity. An extended linker will be useful for expansion of the recognition code of zinc finger proteins and for development of a new role for linker sequences in DNA binding of zinc finger proteins.
Nomura
Wataru
W
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Sugiura
Yukio
Y
D019943:Amino Acid Substitution / Q000235:genetics; D018983:DNA Footprinting; D021901:DNA, Intergenic / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods; D016335:Zinc Fingers / Q000235:genetics*
D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D010455:Peptides; D016329:Sp1 Transcription Factor