Missing contact probing of DNA-protein interactions

Abstract

We have examined the positions of contact between lambda phage repressor protein and operator OR1 DNA by scanning populations of lightly depurinated or depyrimidated DNA for bases essential to or irrelevant to repressor binding. This global scanning technique delineates the apparent contact region between lambda repressor and operator and shows bases previously demonstrated or predicted to be contacted plus some additional bases. A mutant repressor, previously shown to contact DNA as wild-type repressor does with the exception of a missing contact to guanosine G4' [Hochschild, A. & Ptashne, M. (1986) Cell 44, 925-933], similarly failed to contact G4' when assayed by this method. Coupled with altering a test residue of a DNA-contacting protein to glycine or alanine so as to eliminate a specific contact, the method appears to provide an efficient means of scanning for specific residue-base contacts.