Interaction of Proteins with Partially Depurinated and Depyrimidinated Oligonucleotides (Missing-Contact Probing of Protein-DNA Interactions)

Abstract

The procedure first reported by Majors (Majors, 1976, cited by Brunelle and Schleif, 1987) and developed by Brunelle and Schleif (1987) involves the binding of a defined protein to a partially depurinated or depyrimidinated end-labelled oligonucleotide (see Fig. 1). From the mixture of modified DNA oligonucleotides, the protein only binds to those where the bases required for the specific amino acid base contact are intact. As seen in Fig. 1, bases 3 and 4 are important for binding of the protein, whereas bases 1, 2 and 5 may be removed without affecting formation of the protein-DNA complex. This situation is reflected in the final PAGE analysis on a sequencing gel. However, meaningful interpretation of the data from such an experiment assumes that the absence of a base causes no significant structural alteration of the DNA. The essential steps involved in the procedure are shown in the flow diagram in Fig. 2. separation of the protein-DNA complex by gel-shift assay, the DNA is extracted from the gel and subjected to ß-elimination with piperidine. The final reaction products are further separated on a polyacrylamide sequencing gel, which is subsequently autoradiographed on an X-ray film.