Cross-linking of DNA with trimethylpsoralen is a probe for chromatin structure

Abstract

Me 3-psoralen (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-me 3-psoralen inside mouse liver nuclei to the extent of one covalently bound me 3-psoralen molecule per 114 or per 246 base pairs (bp) on the average. The photoreacted nuclei were digested with micrococcal nuclease, which is known to degrade the DNA preferentially between nucleosomes (subunits of chromatin containing approximately 200 bp of DNA). The digestion of nuclei with micrococcal nuclease produced DNA fragments of the same molecular weights whether or not the nuclei had been reacted with me 3-psoralen. The rate of acid solubilization of covalently bound 3H-me 3-psoralen was much greater than that of the bulk DNA when the digestion was performed in nuclei, even though me 3-psoralen-containing regions of DNA were digested more slowly in control experiments with deproteinized DNA. At the digestion limit, when 45% of the DNA had been degraded to acid solubility, 92% of the 3H-me 3-psoralen had been released from chromatin by micrococcal nuclease. Throughout the nuclease digestion, the me 3-psoralen was found to be covalently bound to DNA fragments with the molecular weights previously characterized for micrococcal nuclease digestion products. We conclude that the major site of me 3-psoralen reaction in nuclei is with the DNA most susceptible to micrococcal nuclease—that is, with the DNA between nucleosomes. Because me 3-psoralen cross-links can be located in DNA by electron microscopy, this probe provides a possible method for determining the in vivo location of chromosomal proteins along high molecular weight DNA, such as a transcription unit.