ZERO-LENGTH PROTEIN-NUCLEIC ACID CROSS-LINKING BY RADICAL-GENERATING COORDINATION-COMPLEXES AS A PROBE FOR ANALYSIS OF PROTEIN-DNA INTERACTIONS IN-VITRO AND IN-VIVO

Redox-active coordination complexes such as 1,10-phenanthroline-Cu(II) (OP-Cu) and bleomycin-Fe(III) are commonly used as ''chemical nucleas es'' to introduce single-strand breaks in nucleic acids. Here we repor t that under certain conditions these complexes may crosslink proteins to nucleic acids. In vitro experiments suggest that proteins are cros slinked to DNA by a mechanism similar to dimethyl sulfate-induced cros slinking. Furthermore, we demonstrate that the OP-Cu complex can gener ate protein-DNA crosslinks in mammalian cells in vivo. By combining th e OP-Cu crosslinking and a ''protein shadow'' hybridization assay we i dentify proteins interacting with DNA in isolated pea chloroplasts and show that this methodology can be applied to detect DNA-binding prote ins on specific DNA sequences either in vitro or in vivo. (C) 1998 Aca demic Press.