Modular construction of extended DNA recognition surfaces: mutant DNA-binding domains of the 434 repressor as building blocks

Single-chain derivatives of the 434 repressor containing onewild-type and one mutant DNA-binding domain recognize the generaloperator ACAA–6 base pairs–NNNN, where the ACAAoperator subsite is contacted by the wild-type and the NNNNtetramer by the mutant domain. The DNA-binding specificitiesof several single-chain mutants were studied in detail and theoptimal subsites of the mutant domains were determined. Thecharacterized mutant domains were used as building units toobtain homo- and heterodimeric single-chain derivatives. TheDNA-binding properties of these domain-shuffled derivativeswere tested with a series of designed operators of NNNN–6base pairs–NNNN type. It was found that the binding specificitiesof the mutant domains were generally maintained in the new environmentsand the binding affinities for the optimal DNA ligands werehigh (with K_d values in the range of 10^–11–10^–10M). Considering that only certain sequence motifs in place ofthe six base pair spacer can support optimal contacts betweenthe mutant domains and their subsites, the single-chain 434repressor mutants are highly specific for a limited subset of14 base pair long DNA targets.