Location of the primary sites of micrococcal nuclease cleavage on the nucleosome core.
The positions and relative frequencies of the primary cleavages made by micrococcal nuclease on the DNA of nucleosome core particles have been found by fractionating the double-stranded products of digestion and examining their single-stranded compositions. This approach overcomes the problems caused by secondary events such as the exonucleolytic and pseudo-double-stranded actions of the nuclease and, combined with the use of high resolution gel electrophoresis, enables the cutting site positions to be determined with a higher precision than has been achieved hitherto. The micrococcal nuclease primary cleavage sites lie close (on average, within 0.5 nucleotide) to those previously determined by Lutter (1981) for the nucleases DNase I and DNase II. These similarities show that the accessible regions are the same for all three nucleases, the cleavage sites being dictated by the structure of the nucleosome core. The differences in the final products of the digestion are explained in terms of secondary cleavage events of micrococcal nuclease. While the strongly protected regions of the nucleosome core DNA are common to all three nucleases, there are differences in the relative degrees of cutting at the more exposed sites characteristic of the particular enzyme. In particular, micrococcal nuclease shows a marked polarity in the 3'-5' direction in the cutting rates as plotted along a single strand of the nucleosomal DNA. This is explained in terms of the three-dimensional structure of the nucleosome where, in any accessible region of the double helix, the innermost strand is shielded by the outermost strand on the one side and the histone core on the other. The final part of the paper is concerned with the preference of micrococcal nuclease to cleave at (A,T) sequences in chromatin.
D000818:Animals; D001345:Autoradiography; D001482:Base Composition; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis; D004278:DNA, Superhelical / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008836:Micrococcal Nuclease; D009707:Nucleosomes / Q000032:analysis*
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D009707:Nucleosomes; D004247:DNA; D008836:Micrococcal Nuclease