Quantitative analysis of DNase I digestion patterns of oligo- and polynucleosomes

DNase I digestion of unlabelled chromatin has been used to determine the orientation of nucleosomes in the higher-order structure of chromatin. DNA digestion patterns were analysed quantitatively and compared with simulation curves that were generated with the experimentally obtained rate constants for cutting inside nucleosomes and the half-height widths of the bands. The rate constants for cutting at the linker DNA were varied to fit the experimental curves. By comparing the digestion profiles of polynucleosomes, oligonucleosomes and H1/H5-depleted oligonucleosomes we have been able to distinguish between protection caused by H1/H5 histones only and protection caused by the higher-order structure. By the nature of this method the area protected by H1/H5 histones appears symmetrical around the centre of the nucleosomal DNA on the dyad axis (position S[0]), but it can also be interpreted as a superposition of two separate protected areas that are symmetrical around position S[0]. Protection by the higher-order structure shows that the nucleosomes are oriented with their S[0] positions inside the 30 nm fibre and that there is a minimum number of nucleosomes required for this structure to be formed. We have also found that the linker DNA is cut in a continuous (non-periodical) way and that there are considerable amounts of nucleosomes at discrete distances of multiples of ten nucleotides (10a nt) as well as stretches of nucleosomes positioned either at random or at distances of (10a + 5)nt.