DNase I footprinting of the nucleosome in whole nuclei

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Abstract

DNase I was used to footprint the 147 bp DNA fragment of the nucleosome in whole chicken erythrocyte nuclei. It was found that the higher-order structure imposes an additional protection on nucleosomes at sites close to the entry and exit points of the linker DNA, around the dyad axis (site S 0). The observed protection is extended up to 20 bp on either side of S 0. It is partial (∼50%) and most probably reflects a full protection of different regions in alternatively oriented nucleosomes. These are the same regions which interact with linker histones. The results strongly support the findings by simulation of DNase I digests of unlabelled oligonucleosome fragments in the 30 nm fibre that in all nucleosomes sites S −5 to S −3 and S +3 to S +5 ara on the outside of the fibre exposed to DNase I.

Section snippets

Materials and methods

Nuclei. Chicken blood was collected in PBS (0.15 M NaCl, 0.015 M phosphate, pH 7.6) containing 50 mg/l heparin. Cells were never exposed to EDTA. The cells were lysed in 50 mM Tris pH 7.5, 6 mM MgCl2, 0.25 M sucrose and 1% Triton X-100, followed by several washes in the same buffer without Triton as in Ref. [3].

Nucleosome footprints. Nucleosome footprints were obtained as previously described in Ref. [12], but the nuclei were digested with DNase I before isolation of nucleosomes. Briefly, nuclei at

Results and discussion

To obtain a direct footprint of the additional protection imposed by the higher-order chromatin structure on the nucleosome: (i) whole nuclei were digested with DNase I, (ii) nucleosomes were isolated by micrococcal nuclease digestion and sucrose gradient fractionation, (iii) histone tails were removed by partial trypsin digestion, (iv) nucleosomal DNA was trimmed to core particle size and (v) gel purified and 5’-end-labelled DNA fragments were examined on a denaturing polyacrylamide gel. A

Acknowledgments

I thank Dr. Yana Proykova for discussions and critical reading of the manuscript. This work was supported by The Wellcome Trust Grant No. 037008.

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