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 stru cture consisting of independent finger modules. They are expected to elevat e the DNA binding affinity and specificity by increasing the number of fing er modules. To investigate the relation between the number and the DNA bind ing affinity of the zinc finger, we have designed the two- to four-finger p eptides by connecting the central zinc finger (finger 2) of Sp1 with the ca nonical linker sequence, Thr-Gly-Glu-Lys-Pro. Gel mobility shift assays rev eal that the cognate three- and four-finger peptides, Sp1(zf222) and Sp1(zf 2222), strongly bind to the predicted target sequences, but the two-finger peptide, Sp1(zf22), does not. Of special interest is the fact that the diss ociation constant for Spl(zf2222) binding to the target DNA is comparable t o that for Spl(zf222). The methylation interference, DNase I and hydroxyl r adical footprintings, and circular permutation analyses demonstrate that Sp l(zf2222) binds to its target site with three successive zinc fingers and t he binding of the fourth zinc finger is inhibited by DNA bending induced by the binding of the three-finger domain. The present results strongly indic ate 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 f or long DNA sequences, such as chromosomal DNAs.