PROBING DNA - PROTEIN INTERACTIONS INVITRO WITH THE CPG DNA METHYLTRANSFERASE

A sensitive method was devised to monitor the in vitro binding of nucl ear proteins from HeLa cells presumably to the major groove of DNA. Up on the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methyla ted cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associ ated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequ ences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against th e reaction with DNaseI also escaped in vitro methylation by the CpG DN A methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA - protein interactions which are specific for the major groove of DNA.