HYDROXYL RADICAL FOOTPRINTING OF DNA COMPLEXES OF THE ETS DOMAIN OF PU.1 AND ITS COMPARISON TO THE CRYSTAL-STRUCTURE

Hydroxyl radical footprinting has been used to probe interactions in c omplexes between the ets domain of the murine transcription factor PU. 1 and three different DNA restriction fragments, each containing one c opy of the recognition sequence 5'-GGAA-3'. Two natural PU.1 binding s ites, the SV40 enhancer site and the lambda B motif of Ig lambda 2-4 e nhancer, were used as well as the PU.1 binding site present in the cry stallized PU.1-DNA complex [Kodandapani, R., Pio, F., Ni, C.-Z., Picci alli, G., Klemsz, M., McKercher, S. R., Maki, R. A., and Ely, K. R. (1 996) Nature 380, 456-460]. The footprints obtained for the three diffe rent DNA sequences are almost identical. The extent of contact with th e protein was monitored for every base in the complex. Two concentrati on-dependent cleavage sites on the complementary TTCC strand are evide nce of a specific interaction between PU.1 and the DNA. Two more prote ction sites and a hypersensitive cleavage site on the GGAA strand were observed. Although these data confirm the global structure of the PU. 1-DNA complex as suggested by crystallography, the footprinting data r eveal differences between the protein-DNA contacts in solution and in the crystal state. An additional interaction site not present in the c rystal structure was observed by hydroxyl radical footprinting.