Identification of sequence-specific DNA-binding factors by label transfer: application to the adenovirus-2 major late promoter.

A method of affinity labelling proteins specifically associated with DNA target sequences is proposed. The method utilizes covalent UV-crosslinking of proteins to highly labelled DNA (e.g. in crude cell or nuclear extracts) followed by degradation of the DNA to short oligonucleotides. Proteins selectively labelled by attached residual oligonucleotides are readily amenable to molecular mass determination. Using this approach, we have characterized a HeLa polypeptide specifically bound to a short segment of the adenovirus-2 major late promoter (Ad2 MLP). A molecular mass value (approximately 51 kD) and precise location of the crosslinking site(s) of the protein within the MLP (-55 with respect to the cap site) were determined.