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Nucleic Acids Research, Vol 27, Issue 17 3474-3480, Copyright © 1999 by Oxford University Press

ARTICLES

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

A Simoncsits, ML Tjornhammar, S Wang and S Pongor
International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Padriciano 99, I-34012 Trieste, Italy. simoncs@icgeb.trieste.it

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 altered specificities. The library members contain one wild- type DBD and one mutant domain with randomized amino acids in the DNA- contacting region. Based on previous studies, the mutant sc derivatives are expected to recognize a general ACAA-6 bp-NNNN sequence, where ACAA is contacted by the wild-type and 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 was used to isolate mutant sc repressors that interact with an asymmetric operator 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 studies revealed that certain mutants bound with substantially higher affinities (K(d) approximately 10(-11)M) to operators containing the TTAC sequence, a close homolog of the TTAA target. Thus, we have fortuitously isolated mutant sc repressors that show up to a several hundred-fold preference for TTAC over TTGT.
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 Protein Eng Des SelHome page
T. Liang, J. Chen, M.-L. Tjornhammar, S. Pongor, and A. Simoncsits
Modular construction of extended DNA recognition surfaces: mutant DNA-binding domains of the 434 repressor as building blocks
Protein Eng. Des. Sel., August 1, 2001; 14(8): 591 - 599.
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