Precise location of DNase I cutting sites in the nucleosome core determined by high resolution gel electrophoresis

Abstract

The precise locations of the DNase I cutting sites in the nucleosome core have been determined by analysis of the DNA products of a DNase I digestion of 32p end-labelled nucleosome cores on a high resolution gel electrophoresis system. This system is capable of resolving fragments of mixed sequence DNA differing by one base into the region of 160 bases in length. The DNase I cutting sites in the core are found to be spaced at multiples of about 10.4 (i.e. clearly different from 10.0) bases along the DNA, but show significant variations about this value. In addition to the location of the sites, the stagger between individual sites on opposite strands has been determined and is found to be inconsistent with at least one proposed mechanism for nuclease cleavage of chromatin DNA. Finally, a calculated distribution of fragment lengths in a DNase I digest of nuclei has been determined from the data obtained from the nucleosome core and found to be in reasonable agreement with the observed distribution. The periodicity of 10.4 is discussed with respect to the number of base pairs per turn of chromatin DNA and the number of superhelical turns of DNA per nuc leosome.

The helical periodicity of DNA on the nucleosome

“…and S12W, or Slc and S13w, with staggers varying from about 1.4 to about 3.0 bases (8). This variability of staggers means, as discussed earlier (8),…”

“…The locations of DNAse I cleavage sites along one strand of the nucleosomal DNA have been determined using high resolution gel electrophoresis to measure the lengths of the single-stranded DNA fragments produced by the action of DNAse I on 5'-terminal radioactively labelled nucleosome cores (8).…”

“…2). Now from Lutter's data (8) we have the information from which to place these sites relative to the dyad axis: they are staggered with respect to each other by 1.4 bases in a known direction, and so are each 0.7 bases away from the dyad, along their respective strands.…”

“…Ironically, however, one of the quantities used in making the prediction, namely the screw of DNA on the nucleosome, was shortly thereafter called into question by more accurate measurements, made by ourselves and our colleagues, of the average distance between DNAse I cutting sites of the DNA in nuclei, which give a value of about 10.4 base pairs per turn, rather than 10 (7,8).…”

Sequence-dependent Nucleosome Structural and Dynamic Polymorphism. Potential Involvement of Histone H2B N-Terminal Tail Proximal Domain

“…Sequencing gels were prepared according to standard protocols, except that the acrylamide/bisacrylamide weight ratio was decreased from the usual 19:1 value to 12:1, to better resolve mixed-sequence fragments. (We failed to decrease that ratio to 6 -8:1, as described, 23,24 because the gel would not stick onto the glass plates. )…”