Base-pair resolution mapping of nucleosome positions using site-directed hydroxy radicals

Abstract

This chapter discusses the base-pair resolution mapping of nucleosome positions using site-directed hydroxy radicals. The site-directed hydroxyl radical method presented in the chapter differs from free-solution hydroxyl radical methods because it localizes the chelation of the hydroxy radical-generating iron center to a specific cysteine residue, thus directing the site of attack of hydroxy radicals. Using the chelating EDTAcyst reagent, which is attached via a disulfide bond to histone H4 residue 47 located near the nucleosome pseudo dyad axis, hydroxy radicals create a single major scission per strand separated by three base pairs on complementary strands. Similar site-directed chelating reagents have also been synthesized, and there are growing numbers of reports of their use in the study of nucleic acid interactions. The chapter describes the assembly of nucleosomes containing a derivatized histone octamer.