MAPPING NUCLEOSOME POSITION AT SINGLE BASE-PAIR RESOLUTION BY USING SITE-DIRECTED HYDROXYL RADICALS

A base-pair resolution method for determining nucleosome position in v itro has been developed to complement existing, less accurate methods. Cysteaminyl EDTA ,vas tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specificall y by hydroxyl radical-catalyzed chain scission by using the Fenton rea ction. Strand cleavage occurs mainly at a single nucleotide close to t he dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA, The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegat us 5S rRNA sequence and a twofold-symmetric derivative. The weakness o f translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demo nstrated. The predominant location of both histone octamer and H3H4 te tramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucl eosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptab le for determining nucleosome position in chromatin in vivo.