Engineering of DNA binding proteins into site-specific cutters: Reactivity of Trp repressor-1,10-phenanthroline chimeras

Ralf Landgraf, Clark Pan, Christopher Sutton, Lori Pearson, David S. Sigman

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Trp repressor (TrpR) can be converted into a site-specific nuclease by chemical modification of the cysteine mutants TrpR D46C or TrpR E49C with 5-iodoacetamido-1,10-phenanthroline (OP). In the presence of cupric ion and 3-mercaptopropionic acid, TrpR-regulated operators are cleaved. The properties of these semisynthetic scission reagents have been compared. The E49C construct cleaves efficiently at two sites within the operator and the D46C cleaves at multiple sites. Molecular modeling indicates that the reason for the focused reactivity of E49C is that the OP is rigidly oriented in the protein-DNA complexes whereas the OP can adopt several orientations in TrpR D46C. Mutations and reaction conditions that increase the affinity of the repressor enhance the scission efficiency which approaches 100% within the acrylamide matrix. TrpR E49C-OP smoothly cleaves the trpEDCBA operator in a plasmid in a reaction dependent on the corepressor L-tryptophan. In the absence of tryptophan, non-specific cleavage of the plasmid is observed under the same conditions. Therefore, tryptophan not only directs cleavage to a specific site but also blocks it at non-specific sites. The analysis of the cleavage pattern of the trpEDCBA operator provides strong evidence for the tandem binding model in which protein-protein interactions stabilize binding on the DNA. TrpR E49C-OP should serve as the basis for the engineering of a family of highly specific semisynthetic scission reagents.

Original languageEnglish
Pages (from-to)603-610
Number of pages8
JournalProtein Engineering
Volume9
Issue number7
StatePublished - Jul 1 1996
Externally publishedYes

Fingerprint

DNA-Binding Proteins
DNA
Proteins
Tryptophan
Molecular modeling
Chemical modification
Plasmids
3-Mercaptopropionic Acid
Acids
Ions
Co-Repressor Proteins
Acrylamide
E coli TRPR protein
1,10-phenanthroline
Carrier Proteins
Cysteine
Mutation

Keywords

  • Binding proteins
  • Reactivity
  • Site-specific cutters
  • Trp repressor-1,10-phenanthroline chimeras

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Engineering of DNA binding proteins into site-specific cutters : Reactivity of Trp repressor-1,10-phenanthroline chimeras. / Landgraf, Ralf; Pan, Clark; Sutton, Christopher; Pearson, Lori; Sigman, David S.

In: Protein Engineering, Vol. 9, No. 7, 01.07.1996, p. 603-610.

Research output: Contribution to journalArticle

Landgraf, Ralf ; Pan, Clark ; Sutton, Christopher ; Pearson, Lori ; Sigman, David S. / Engineering of DNA binding proteins into site-specific cutters : Reactivity of Trp repressor-1,10-phenanthroline chimeras. In: Protein Engineering. 1996 ; Vol. 9, No. 7. pp. 603-610.
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