Design and synthesis of sequence-specific DNA-binding peptides

Design, synthesis and DNA binding activities of two peptides containing 32 and 102 residues are reported. A nonlinear 102-residue peptide contains four modified alpha helix-turn-alpha helix motifs of 434 cro protein. These four units are linked covalently to a carboxyterminal crosslinker containing four arms each ending with an aliphatic amino group. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha-helical, beta-sheet and random-coiled conformations with the alpha-helical content of about 16% at room temperature. Upon complex formation between peptide and DNA, a change in the peptide conformation takes place which is consistent with an alpha - beta transition in the DNA binding alpha helix-turn-alpha helix units of the peptide. Similar conformation changes are observed upon complex formation with the synthetic operator of a linear peptide containing residues 7-37 of 434 cro repressor. Evidently, in the complex, residues present in helices alpha 2 and alpha 3 of the two helix motif form a beta-hairpin which is inserted in the minor DNA groove. The last inference is supported by our observations that the two peptides can displace the minor groove-binding antibiotic distamycin A from poly(dA).poly(dT) and synthetic operator DNA. As revealed from DNase digestion studies, the nonlinear peptide binds more strongly to a pseudooperator Op1, located in the cro gene, than to the operator OR3. A difference in the specificity shown by the non-linear peptide and wild-type cro could be attributed to a flexibility of the linker chains between the DNA-binding domains in the peptide molecule as well as to a replacement of Thr-Ala in the peptide alpha 2-helices. Removal of two residues from the N-terminus of helix alpha 2 in each of the four DNA-binding domains of the peptide leads to a loss of binding specificity.     

Accessibility of DNA-epoxycellulose to sequence-specific DNA-binding proteins.

Phage lambda DNA was covalently coupled to epoxy-activated cellulose to form a stable DNA-cellulose matrix for affinity chromatography of sequence-specific DNA-binding proteins. The accessibility of three specific six-base sequences, GGATCC (BamHI), GAATTC (EcoRI) and AAGCTT (HindIII) was studied quantitatively and qualitatively by restriction analysis followed by labelling of their recessed ends. All sites are randomly accessible. The site accessibility is variable, BamHI greater than HindIII greater than EcoRI, and within the range 20-100% depending on base composition and internal structure of the sequence. DNA-epoxycellulose, because of its high efficiency of coupling, capacity, stability and accessibility, can be of great help in the isolation and characterization of sequence-specific DNA-binding proteins.