Reconstruction of complexes of histone and superhelical nuclear dna

• DOI: 10.1242/jcs.50.1.209
• Publication Date: 2021-04-27T01:21:54Z
• Authors: Jeremy M. Levin; Peter R. Cook
• Keywords: Histones; 0301 basic medicine; 03 medical and health sciences; Spectrometry, Fluorescence; DNA, Superhelical; Humans; Micrococcal Nuclease; Nucleic Acid Conformation; Microscopy, Phase-Contrast; Polylysine; Sodium Chloride; HeLa Cells

ABSTRACT When HeLa cells are lysed in solutions containing a non-ionic detergent and 2 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear RNA and DNA but few of the proteins characteristic of chromatin. Their DNA is supercoiled and so intact. Using a simple and rapid procedure we have reconstructed nucleohistone complexes from nucleoids and the ‘core’ histones without breaking the DNA. We have probed the integrity and structure of the reconstructed complexes using a non-destructive fluorometric approach, which provides a general method for detecting agents that bind to DNA and alter its supercoiling. The βuperhelical status of the DNA in the reconstructed complexes is indistinguishable from that found in control nucleoids containing core histones. Experiments with micrococcal nuclease confirm that the DNA in the reconstructed complexes is organized into nucleosome-like structures. These, however, are spaced 145 base-pairs apart and not 200 base-pairs apart as is found in native chromatin.