Jupe E R, Sinden R R, Cartwright I L
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, OH 45267-0524.
EMBO J. 1993 Mar;12(3):1067-75. doi: 10.1002/j.1460-2075.1993.tb05748.x.
A psoralen crosslinking assay was utilized to detect localized, unrestrained DNA supercoiling (torsional tension) in vivo in Drosophila chromosomal regions subject to differential transcriptional activity. By comparing rates of crosslinking in intact cells with those in cells where potential tension in chromosomal domains was relaxed by DNA strand nicking, the contribution to psoralen accessibility caused by altered DNA-protein interactions (e.g. nucleosomal perturbations) was distinguished from that due to the presence of unrestrained supercoiling in a region of interest. The heat shock protein 70 (hsp70) genes were wound with a significant level of superhelical tension that remained virtually unaltered whether or not the genes were transcriptionally activated by thermal elevation. Constitutively expressed 18S ribosomal RNA genes also exhibited unrestrained superhelical tension at a level comparable with that across hsp70. In contrast, flanking regions downstream of each of the divergent hsp70 genes at locus 87A7 exhibited substantially less tension. Thus the results point to the existence of stable, torsionally stressed topological domains within eukaryotic chromosomal DNA, suggesting that the relaxing action of topoisomerases is not ubiquitous throughout the nucleus but, in fact, is likely to be tightly regulated.
采用补骨脂素交联试验来检测果蝇染色体区域中体内局部不受限制的DNA超螺旋（扭转张力），这些区域具有不同的转录活性。通过比较完整细胞中的交联速率与染色体结构域中潜在张力通过DNA链切口得以松弛的细胞中的交联速率，区分了由DNA-蛋白质相互作用改变（如核小体扰动）导致的补骨脂素可及性变化与由于感兴趣区域中存在不受限制的超螺旋而导致的补骨脂素可及性变化。热休克蛋白70（hsp70）基因缠绕着显著水平的超螺旋张力，无论这些基因是否通过热激激活转录，该张力基本保持不变。组成型表达的18S核糖体RNA基因也表现出不受限制的超螺旋张力，其水平与hsp70基因的相当。相比之下，位于87A7位点的每个双向hsp70基因下游的侧翼区域表现出的张力要小得多。因此，结果表明真核染色体DNA中存在稳定的、受到扭转应力的拓扑结构域，这表明拓扑异构酶的松弛作用在整个细胞核中并非普遍存在，实际上可能受到严格调控。
