DNase I was used to footprint the 147 bp DNA fragment of the nucleosome in whole chicken erythrocyte nuclei. It was found that the higher-order structure imposes an additional protection on nucleosomes at sites close to the entry and exit points of the linker DNA, around the dyad axis (site S 0). The observed protection is extended up to 20 bp on either side of S 0. It is partial (similar to 50%) and most probably reflects a full protection of different regions in alternatively oriented nucleosomes. These are the same regions which interact with linker histones. The results strongly support the findings by simulation of DNase 1 digests of unlabelled oligonucleosome fragments in the 30 nm fibre that in all nucleosomes sites S -5 to S -3 and S +3 to S +5 ara on the outside of the fibre exposed to DNase I. (C) 2008 Elsevier Inc. All rights reserved.