Design superiority of palindromic DNA sites for site-specific recognition of proteins: Tests using protein stitchery

Using protein stitchery with appropriate attachment of cysteines linking to either C or N termini of the basic region of the v-Jun leucine zipper gene-regulatory protein, we constructed three dimers - pCC, pCN, and pNN. All three bind specifically to the appropriately rearranged DNA recognition sites for v-Jun: ATGAcgTCAT, ATGAcgATGA, and TCATcgTCAT, respectively (K_d, ≈4 nM at 4°C). Results of DNase I footprinting provide strong support for bent recognition helices in leucine zipper protein-DNA complexes. Comparison of the results for pCC and pNN with those for pCN shows the design superiority of palindromic sequences for protein recognition.