Isolation of altered specificity mutants of the single-chain 434 repressorthat recognize asymmetric DNA sequences containing the TTAA and TTAC subsites

A novel single-chain (sc) protein framework containing covalently dimerized DNA-binding domains (DBD) of the phage 434 repressor was used to construct combinatorial mutant libraries in order to isolate mutant DBDs with altere d specificities. The library members contain one wild-type DBD and one muta nt domain with randomized amino acids in the DNA-contacting region, Based o n previous studies, the mutant sc derivatives are expected to recognize a g eneral ACAA-6 bp-NNNN sequence, where ACAA is contacted by the wild-type an d NNNN by the mutant domain, In principle, any sequence can stand for NNNN and serve as a selection target, Here an in vivo library screening method w as used to isolate mutant sc repressors that interact with an asymmetric op erator containing the TTAA target, Several mutants showed high affinity in vitro binding to operators containing the target and strong (up to 80-fold) preference for the TTAA target over the wild-type TTGT, Specificity studie s revealed that certain mutants bound with substantially higher affinities (K-d similar to 10(-11) M) to operators containing the TTAC sequence, a clo se homolog of the TTAA target. Thus, we have fortuitously isolated mutant s c repressors that show up to a several hundredfold preference for TTAC over TTGT.