Selective Nucleosome Disruption by Drugs That Bind in the Minor Groove of DNA

Daniel J Fitzgerald,John N Anderson

doi:10.1074/jbc.274.38.27128

Abstract

Previous studies have shown that drugs which bind in the DNA minor groove reduce the curvature of bent DNA. In this article, we examined the effects of these drugs on the nucleosome assembly of DNA molecules that display different degrees of intrinsic curvature. DAPI (4,6-diamidino-2-phenylindole) inhibited the assembly of a histone octamer onto a 192-base pair circular DNA fragment from Caenorhabditis elegans and destabilized a nucleosome that was previously assembled on this segment. The inhibitory effect was highly selective since it was not seen with nonbent molecules, bent molecules with noncircular shapes, or total genomic DNA. This marked template specificity was attributed to the binding of the ligand to multiple oligo A-tracts distributed over the length of the fragment. A likely mechanism for the effect is that the bound ligand prevents the further compression of the DNA into the minor groove which is required for assembly of DNA into nucleosomes. To further characterize the effects of the drug on chromatin formation, a nucleosome was assembled onto a 322-base pair DNA fragment that contained the circular element and a flanking nonbent segment of DNA. The position of the nucleosome along the fragment was then determined using a variety of nuclease probes including exonuclease III, micrococcal nuclease, DNase I, and restriction enzymes. The results of these studies revealed that the nucleosome was preferentially positioned along the circular element in the absence of DAPI but assembled onto the nonbent flanking sequence in the presence of the drug. DAPI also induced the directional movement of the nucleosome from the circular element onto the nonbent flanking sequence when a nucleosome preassembled onto this template was exposed to the drug under physiologically relevant conditions.

Highlights

The nucleosome is the basic structural unit of chromatin
Little is known about selective changes in chromatin structure that might occur upon drug binding, such changes are likely to be important in the mechanism of action of these compounds
Effects of Minor Groove Binding Drugs on Nucleosome Assembly—The studies described below focus on a DNA segment from an intron of the C. elegans RNA polymerase II gene [33, 40]

Summary

EXPERIMENTAL PROCEDURES

DNA Preparation and Characterization—Standard molecular biological techniques were used for DNA manipulations [31]. The high ionic strength electrophoretic analysis described in the text was carried out using an 8% polyacrylamide (PA) gels run for 3 days at 4 °C with buffer recirculation at 30 V in Tris acetate-EDTA(TAE) buffer with or without 0.6 M NaCl. Radioactive DNA was prepared by polymerase chain reaction using primers (25-mers) that are centered at 19 bp upstream and 33 bp downstream from the EcoRI site and HindIII site in pUC18, respectively (see Fig. 1). Restriction enzyme studies, digestions were carried out for 1 h at room temperature in 10 mM MgCl2 containing either 50 or 100 mM NaCl and the fragments analyzed on 8% native PA gels. Binding at all temperatures as determined by fluorescence enhancement at 465 nM was essentially maximum within 3 min with both nucleosome and naked DNA and binding extents were similar with naked Vs corresponding nucleosome DNA

RESULTS

Selective Nucleosome Disruption

The studies of the nucleosomes assembled onto fragments

DISCUSSION