DNA recognition by Zn [Cysteinyl-His-OMe]₂: a minimal zinc finger docking unit

Abstract

Zinc fingers, 30-residue peptides anchored on Zn(II) coordinated to pairs of cysteines and histidines, recognize DNA triplets and, as tandem modules, effect sequence read out. The focus of zinc finger-DNA interaction studies thus far has been to probe the nature of the binding of the 12-residue recognition element of the finger with DNA code bases. To understand the possible role of the Zn(II) ligand and to assess its own DNA interaction profile, [(CH)₂Zn] (C: cysteine; H: histidine; Figure 1) was constructed from bis^-t Boc-cystinyl-di-His-OMe via thiol-disulfide exchange, Zn(II) complexation, and deprotection. [(CH)₂Zn] binds with polyd- (GC)polyd(GC) with association constants-1.8 × 10⁷ M^-1 (specific DNA-phosphate) and 3.3 × 10³ M^-1 (nonspecific DNA-phosphate); perturbs its B-DNA profile; and enhances the T_m from 62.5 to 70.15° C in a concentration-independent manner, with an ideal reversal profile on cooling, not observed in the DNA alone; releases polyd(GC)polyd(GC)-bound ethidium bromide; enhances the fluorescence of polyd(GC)polyd(GC)-bound ethidium bromide at low concentrations; and quenches it at higher ranges. [(CH)₂Zn] also binds to d(ACGCTGGGCGT), the sequence associated with Zif-268, 3-finger binding site. Such interactions were not seen in parallel studies with (a) polyd(AT)polyd(AT) and [(CH)₂Zn] and (b) {[C'H₂] (C': cystine; H: histidine; the direct metal-free precursor of [(CH)₂Zn]}, ionic zinc nitrate, and covalent zinc acetyl acetonate Zn(AcAc)₂, with poly[d(GC)polyd(GC)]. The results are rationalized on the basis of two types of association between [(CH)₂Zn] and polyd(GC)polyd(GC), a nonspecific recognition of the sugar phosphate backbone, by an imidazole of [(CH)₂Zn] and a specific one involving the amino group of [(CH)₂Zn] and the guanine base of DNA. Control experiments show that the latter greatly promotes DNA recognition. The possibility for such specific interactions with relatively small structures of the type [(CH)₂Zn] would be of use in the design of DNA recognition elements and also provide an explanation for the experimentally found variation in the placement of the zinc finger docking unit around the major groove of DNA.