Multiconnection of identical zinc finger: implication for DNA binding affinity and unit modulation of the three zinc finger domain.
Cys(2)-His(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules. They are expected to elevate the DNA binding affinity and specificity by increasing the number of finger modules. To investigate the relation between the number and the DNA binding affinity of the zinc finger, we have designed the two- to four-finger peptides by connecting the central zinc finger (finger 2) of Sp1 with the canonical linker sequence, Thr-Gly-Glu-Lys-Pro. Gel mobility shift assays reveal that the cognate three- and four-finger peptides, Sp1(zf222) and Sp1(zf2222), strongly bind to the predicted target sequences, but the two-finger peptide, Sp1(zf22), does not. Of special interest is the fact that the dissociation constant for Sp1(zf2222) binding to the target DNA is comparable to that for Sp1(zf222). The methylation interference, DNase I and hydroxyl radical footprintings, and circular permutation analyses demonstrate that Sp1(zf2222) binds to its target site with three successive zinc fingers and the binding of the fourth zinc finger is inhibited by DNA bending induced by the binding of the three-finger domain. The present results strongly indicate that the zinc finger protein binds to DNA by the three-finger domain as one binding unit. In addition, this information provides the basis for the design of a novel multifinger protein with high affinity and specificity for long DNA sequences, such as chromosomal DNAs.
Nagaoka
M
M
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Kaji
T
T
Imanishi
M
M
Hori
Y
Y
Nomura
W
W
Sugiura
Y
Y
D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D017665:Hydroxyl Radical; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D016329:Sp1 Transcription Factor; D017665:Hydroxyl Radical; D004247:DNA; D003850:Deoxyribonuclease I