<i>In vitro</i> drug-induced cleavage in the <i>Drosophila</i> histone-gene repeat.

<p>(A) Schematic representation of the 5-kbp major histone-gene locus, which contains the 5 genes encoding core and linker histones. The bar with a hook represents the scaffold-associated region (SAR) located in the intergenic spacer between the H1 and H3 genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Mirkovitch1" target="_blank">[74]</a>. It is cleaved by Topo II (denoted by scissors) in the linker regions of 5 positioned nucleosomes (black circles). Other sites (1, 2 and 8–10) map to DNAse I-hypersensitive sites at the 5′ and 3′ ends of the histone genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Ks1" target="_blank">[21]</a> for details). Sites 3-5 mapped at nucleotide resolution by direct genomic sequencing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Ks1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023597#pone.0023597-Borgnetto1" target="_blank">[22]</a>. (B) <i>In vitro</i> cleavage induced by teniposide (VM26) and F14512. A uniquely end-labeled DNA clone spanning the histone-gene repeat was incubated with Topo II as described in Materials and Methods in the presence of no drug (lane 1), 50 µM teniposide with 0, 5, 10 or 25 µM distamycin (lanes 5, 4, 3 and 2, respectively) or 5 µM F14512 with 0, 5, 10 or 25 µM distamycin (lanes 6, 7, 8 and 9, respectively). The 4 lanes at the right of the gel are Maxam-Gilbert sequencing reactions of the labeled fragment. Cleavage is induced at numerous identical sites by teniposide and F14512 in the absence of distamycin (compare lanes 5 and 6). Increasing concentrations of distamycin redirect cleavage induced by both drugs to sites 3–5 marked by arrows to the left of the gel and which correspond exactly to the sites cleaved <i>in cellulo</i>.