Electron Microscopy of DNA Cross-linked with Trimethylpsoralen: A Probe for Chromatin Structure

Thomas Cech, David Potter, Mary Lu Pardue

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Abstract

Me3psoralen (4,5′,8-trimethylpsoralen) undergoes a photochemical reaction with DNA, resulting in the formation of covalent monoadducts and interstrand cross-links. DNA was photoreacted with [3H]Me3 psoralen inside mouse liver nuclei to the extent of one covalently bound Me3 psoralen molecule per 114 or per 246 base pairs on the average. DNA purified from these nuclei was prepared for electron microscopy under totally denaturing conditions. Adjacent cross-links in the DNA were found to be separated by intervals of 190 base pairs, or by integral multiples of that length. Deproteinized mouse DNA was also photoreacted with [3H]Me3 psoralen under the same conditions. Electron microscopic measurements of the distances between adjacent cross-links in this DNA did not give the discrete distribution seen in the DNA photoreacted in nuclei. Instead, the pattern of cross-links was consistent with a random distribution of Me3 psoralen reaction sites in the DNA. The Me3 psoralen photoreaction of DNA in intact mouse sperm (where protamines have replaced histones) also resulted in an approximately random distribution of cross-links. The discrete cross-linking pattern generated by the photoreaction of DNA in mouse nuclei therefore seems to reflect the periodicity of proteins in chromatin (the nucleosome subunits), as previously suggested by C. V. Hanson et al. [(1976), Science 193, 62-64]. These methods should allow the mapping of the in vivo location of nucleosomes on transcriptionally active and inactive DNA and on newly replicated DNA.

Original languageEnglish (US)
Pages (from-to)5313-5321
Number of pages9
JournalBiochemistry
Volume16
Issue number24
DOIs
StatePublished - Nov 1 1977

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