High-specific-activity probes and a high-resolution in-gel photo cross-linking assay for protein–DNA complexes

Photochemical cross-linking has been widely employed to identify proteins interacting with specific sites on DNA. Identification of bound proteins usually relies on transfer of a radiolabel from the DNA to the protein by cross-linking. We set out to fine-map a small viral replication preinitiation complex composed of two protein dimers bound to DNA, the bovine papillomavirus E1E2–ori complex. Here we describe a simple method for generating high-specific-activity probes with a phenyl-azide photoactivatible cross-linking group positioned immediately adjacent to a labeled nucleotide. The method is based on the selective destruction of one 5′-phosphorylated strand of a polymerase chain reaction product with λ exonuclease and reconstitution of the probe with a phosphorothioate-substituted oligonucleotide, an [α-32P]dNTP, and thermophilic enzymes. We also developed a high-resolution in-gel cross-linking assay to probe defined protein–DNA complexes. With these methods we have obtained structural information for the papillomavirus E1E2–ori preinitiation complex that would otherwise have been hard to obtain. These approaches should be widely applicable to the study of protein–DNA complexes.