Measurement of unrestrained negative supercoiling and topological domain size in living human cells.
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abstract
Unrestrained DNA supercoiling was measured using a Me3-psoralen photobinding assay within a transcriptionally active hygromycin B phosphotransferase (hph) gene integrated into different chromosomal locations in five transformed human fibrosarcoma cell lines. The level of unrestrained supercoiling in the hph gene varied, from high to low levels, in different chromosomal locations in living human cells. In one cell line, the hph gene contained no unrestrained supercoiling. Consequently, supercoiling was not dictated by the DNA sequence of the active hph gene. The addition of alpha-amanitin, which can inhibit transcription, reduced unrestrained supercoiling by 75% at one chromosomal location, by 50% at two other locations, and had little, if any, effect at two other chromosomal locations. Different levels of supercoiling in separate regions of the chromosome require that the chromosome be organized into independent topological domains in vivo. Evidence for independent topological domains in living cells is presented. From analysis of the relaxation of supercoiling as a function of the number of breaks introduced into the chromosome, the in vivo topological domain size for the human ribosomal RNA genes was estimated between 30,000 and 45,000 kb.
