MASS-SPECTROMETRIC CHARACTERIZATION OF SEQUENCE-SPECIFIC COMPLEXES OFDNA AND TRANSCRIPTION FACTOR PU.1 DNA-BINDING DOMAIN

Electrospray ionization mass spectrometry (ESI-MS) has been used to st udy the noncovalent interaction of the 13.5-kDa DNA binding domain of PU.1 (PU.1-DBD) with specific double-stranded DNA (dsDNA) target molec ules. Mixtures of PU.1-DBD protein and wildtype target DNA sequence yi elded ESI-MS spectra showing only protein-dsDNA complex ions of 1:1 st oichiometry and free dsDNA. When PU.1-DBD protein, wild type target DN A, and a mutant target DNA lacking the consensus sequence were mixed, only the 1:1 complex with the wild-type DNA was observed, consistent w ith gel electrophoresis mobility shift assay results, demonstrating th e observation of sequence-specific protein-dsDNA complexes using ESI-M S. (C) 1996 Academic Press, Inc.