Zero-Length Protein–Nucleic Acid Crosslinking by Radical-Generating Coordination Complexes as a Probe for Analysis of Protein–DNA Interactionsin Vitroandin Vivo

Redox-active coordination complexes such as 1,10-phenanthroline–Cu(II) (OP–Cu) and bleomycin–Fe(III) are commonly used as “chemical nucleases” to introduce single-strand breaks in nucleic acids. Here we report that under certain conditions these complexes may crosslink proteins to nucleic acids.In vitroexperiments suggest that proteins are crosslinked to DNA by a mechanism similar to dimethyl sulfate-induced crosslinking. Furthermore, we demonstrate that the OP–Cu complex can generate protein–DNA crosslinks in mammalian cellsin vivo.By combining the OP–Cu crosslinking and a “protein shadow” hybridization assay we identify proteins interacting with DNA in isolated pea chloroplasts and show that this methodology can be applied to detect DNA-binding proteins on specific DNA sequences eitherin vitroorin vivo.