FOOTPRINTING PROTEINS ON DNA WITH PEROXONITROUS ACID

The peroxonitrite anion (ONOO-) is a stable species in alkaline soluti on that quickly generates a strong oxidant at neutral pH. A convenient procedure for the preparation of ONOOK has been developed based on th e procedure of Keith & Powell [(1969) J. Chem. Soc. A, 90], which when added to a sample of duplex DNA buffered at neutral pH rapidly genera tes a strong oxidant capable of nonspecifically cleaving the DNA prese nt. We show that this solution containing ONOOK can be used to hydroxy l radical footprint the binding the cI-repressor (cI) of phage lambda with the right operator, O(R). In addition, we show that the individua l-site binding isotherms determined by quantitative DNase I, Fe-EDTA a nd ONOOK footprinting are identical within experimental error. The ide ntical isotherms obtained with the three different reagents with great ly differing sampling times indicates that the sampling time of the fo otprinting probe need not be short relative to the kinetic dissociatio n constants that govern protein-DNA interactions.