DNA-protein cross-linking applications for chromatin studies in vitro and in vivo

Covalent protein–nucleic acid cross-linking is a powerful tool for the analysis of protein–DNA and protein–RNA interactions in various biological systems. It allows a researcher to freeze protein–nucleic acid contacts within the context of a nucleoprotein complex and analyze cross-linked species by a variety of methods such as two-dimensional (2D) denaturing gel electrophoresis This chapter describes some of the methodical advances in the field of protein–nucleic acid covalent cross-linking applications. The innovations discussed include (1) a fast, relatively noninvasive method of cross-linking by radical-producing chemicals, such as bleomycin–iron and phenanthroline–copper complexes, (2) applications of chemical and photochemical cross-linking techniques to nucleosome-containing complexes following in vitro nucleosome reconstitution on specific DNA, and (3) a time-resolved cross-linking technique for studies of postreplicative chromatin assembly based on the immunoenrichment of newly replicated DNA. The chapter describes 5S rRNA gene nucleosome interactions with linker histones and transcription factor TFIIIA.