Advancing Epigenetics Towards Systems Biology

Site-directed hydroxy radical mapping of nucleosome positions in vitro (Prot 21)

Andrew Flaus, Chris Stockdale, Elisa Garcia-Wilson, Maik Engeholm, Helder Ferreira, & Tom Owen-Hughes

Introduction

The dynamics of chromatin structure is becoming an area of increasing interest. Both thermal energy and ATP-dependent chromatin remodelling enzymes can alter nucleosome structure and positioning. In addition, modifications to both the DNA and histones and changes in their composition can influence the rate of these changes. The ability to precisely determine the position of a nucleosome in vitro is extremely useful when studying the biochemical behaviour of chromatin and its modifying enzymes.

This protocol gives details of a method for mapping the position of nucleosomes in vitro by attaching an EDTA-derived reagent to a specific site on the histone octamer which then catalyses local DNA cleavage. The sites of cleavage reveal the position of the nucleosome at base pair resolution (Flaus et al., 1996; Flaus and Richmond, 1999a; Flaus and Richmond, 1999b; Bruno et al., 2004). Nucleosome positions and movements on DNA fragments of some 500 bp have been mapped (Flaus and Owen-Hughes, 2003).

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Andrew Flaus, Chris Stockdale, Elisa Garcia-Wilson, Maik Engeholm, Helder Ferreira, & Tom Owen-Hughes

Department of Biochemistry - National University of Ireland Galway - Distillery Road - Galway, Ireland

Andrew Flaus