A zinc finger directory for high-affinity DNA recognition.
We have used two monovalent phage display libraries containing variants of the Zif268 DNA-binding domain to obtain families of zinc fingers that bind to alterations in the last 4 bp of the DNA sequence of the Zif268 consensus operator, GCG TGGGCG. Affinity selection was performed by altering the Zif268 operator three base pairs at a time, and simultaneously selecting for sets of 16 related DNA sequences. In this way, only four experiments were required to select for all possible 64 combinations of DNA triplet sequences. The results show that (i) for high-affinity DNA binding in the range observed for the Zif268 wild-type complex (Kd = 0.5-5 nM), finger 1 specifically requires the arginine at the carboxy terminus of its recognition helix that forms a bidentate hydrogen-bond with the guanine base (G) in the crystal structure of Zif268 complexed to its DNA operator sequence GCG TGG GCG; (ii) when the guanine base (G) is replaced by A, C, or T, a lower-affinity family (Kd > or = 50 nM) can be detected that shows an overall tendency to bind G-rich DNA; (iii) the residues at position 2 on the finger 2 recognition helix do not appear to interact strongly with the complementary 5' base in the finger 1 binding site; and (iv) unexpected substitutions at the amino terminus of finger 1 can occasionally result in specificity for the 3' base in the finger 1 binding site. A DNA recognition directory was constructed for high-affinity zinc fingers that recognize all three bases in a DNA triplet for seven sequences of the type GNN. Similar approaches may be applied to other zinc fingers to broaden the scope of the directory.
Department of Chemistry, University of California, Berkeley 94720, USA.
A C
AC
Jamieson
H
H
Wang
S H
SH
Kim
D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015723:Gene Library; D007700:Kinetics; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; D001120:Arginine