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 one wild-type and one mutant DNA- binding domain recognize the general operator ACAA-6 base p airs-NNNN, where the ACAA operator subsite is contacted by the wildtype and the NNNN tetramer by the mutant domain. The DNA-binding specificities of s everal single-chain mutants were studied in detail and the optimal subsites of the mutant domains were determined. The characterized mutant domains we re used as building units to obtain homo- and heterodimeric single-chain de rivatives. The DNA-binding properties of these domain-shuffled derivatives were tested with a series of designed operators of NNNN-6 base pairs-NNNN t ype. It was found that the binding specificities of the mutant domains were generally maintained in the new environments and the binding affinities fo r the optimal DNA ligands were high (with Kd values in the range of 10(-11) -10(-10) M). Considering that only certain sequence motifs in place of the six base pair spacer can support optimal contacts between the mutant domain s and their subsites, the single-chain 434 repressor mutants are highly spe cific for a limited subset of 14 base pair long DNA targets.