QUANTITATIVE-ANALYSIS OF DNASE I DIGESTION PATTERNS OF OLIGONUCLEOSOMES AND POLYNUCLEOSOMES

DNase I digestion of unlabelled chromatin has been used to determine t he orientation of nucleosomes in the higher-order structure of chromat in. DNA digestion patterns were analysed quantitatively and compared w ith simulation curves that were generated with the experimentally obta ined 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 digestio n profiles of polynucleosomes, oligonucleosomes and H1/H5-depleted oli gonucleosomes we have been able to distinguish between protection caus ed by H1/H5 histones only and protection caused by the higher-order st ructure. By the nature of this method the area protected by H1/H5 hist ones appears symmetrical around the centre of the nucleosomal DNA on t he dyad axis (position S[0]), but it can also be interpreted as a supe rposition 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 f ibre and that there isa minimum number of nucleosomes required for thi s structure to be formed. We have also found that the linker DNA is cu t in a continuous (non-periodical) way and that there are considerable amounts of nucleosomes at discrete distances of multiples of ten nucl eotides (10a nt) as well as stretches of nucleosomes positioned either at random or at distances of (10a + 5)nt. (C) 1998 Academic Press Lim ited.