ELECTROSPRAY-IONIZATION MASS-SPECTROMETRIC CHARACTERIZATION OF PHOTOCROSSLINKED DNA ECORI DNA METHYLTRANSFERASE COMPLEXES

We describe a novel strategy combining photocrosslinking and HPLC-base d electrospray ionization mass spectrometry to identify UV crosslinked DNA-protein complexes, EcoRI DNA methyltransferase modifies the secon d adenine within the recognition sequence GAATTC. Substitution of 5-io douracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5-iodour acil-substituted DNA-protein complex at various wavelengths was optimi zed, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-p rotein complex was further analyzed by electrospray ionization mass sp ectrometry, The total mass is consistent with irradiation-dependent co valent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole q uantities of crosslinked peptides by similar strategies.