ENGINEERING OF DNA-BINDING PROTEINS INTO SITE-SPECIFIC CUTTERS - REACTIVITY OF TRP REPRESSOR-1,10-PHENANTHROLINE CHIMERAS

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 (OF), In the presence of cupr ic ion and 3-mercaptopropionic acid, TrpR-regulated operators are clea ved, 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 modelin g indicates that the reason for the focused reactivity of E49C is that the OP is rigidly oriented in the protein-DNA complexes whereas the O P can adopt several orientations in TrpR D46C. Mutations and reaction conditions that increase the affinity of the repressor enhance the sci ssion efficiency which approaches 100% within the acrylamide matrix, T rpR E49C-OP smoothly cleaves the trpEDCBA operator in a plasmid in a r eaction dependent on the corepressor L-tryptophan. In the absence of t ryptophan, 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 o f the cleavage pattern of the trpEDCBA operator provides strong eviden ce for the tandem binding model in which protein-protein interactions stabilize binding on the DNA, TrpR E49C-OP should serve as the basis f or the engineering of a family of highly specific semisynthetic scissi on reagents.