First, to establish that our hypothesis that transcriptionally generated DNA supercoiling regulates the activity of particular elements, we prepared a model system comprised of an EBV-based episome where the FUSE element was sandwiched between divergent metallothionein promoters. By adding zinc to defined levels we could adjust the level of ongoing transcription and control the forces propagated onto FUSE. Upon exceeding a key threshold, the FUSE element would become single-stranded, recruit the specific single stranded DNA binding protein FBP, and reprogram the output of the metallothionein promoters. Using Cre-recombinase to excise precisely defined DNA circles, allowed us to monitor directly the accompanying changes in DNA topology validating this novel mechanism as potentially general principle to confer precise control on activated promoters. We are now developing experimental and analytical approaches to map the level and distribution of supercoiling forces on a genome-wide basis. to accomplish this we are exploiting the known preference for psoralen to intercalate into negatively supercoiled DNA. Because psoralen readily penetrates living cells and can be cross-linked to DNA by a pulse of UV light, by comparing the relative hybridization of cross-linked to uncross-linked DNA to high density genomic tiling arrays, we can map the genome-wide distribution of psoralen. By including proper controls to account for ongoing transcription, chromatin structure, and by eliminating the influence of replication (by using properly synchronized cells), we can infer the distribution of supercoils. Because high levels of supercoiling promotes the formation of non-B DNA structures, we have also devised a method to map the distribution of non-B DNA across the genome. Virtually any of the non-B DNA structures formed in vitro result in the exposure of DNA bases to the local solvent (at least the the junctions between B and non-B DNA), potassium permanganate can be used as a chemical probe of unpaired bases. Because permanganate rapidly permeates cells, folowing a short exposure to this chemical, when DNA is extracted the permaganate oxidized pyrmidines in stabilize foci of non-B DNA and preserve sensitivity to cleavage by single strand specific endonucleases. Following cleavage with such endonucleases, these ends can be marked, and recovered and analyzed by hybridization to microarrays or by sequencing. Application of this approach reveals enrichment for permanganate sensitive sites in sequences computationally predicted to 1)be sensitive to supercoil induced duplex destabilization (SIDD);2) to be Z-DNA (left handed double helix;or 3) to form G-quadraplex structures. Application of the methods that we have developed reveals the genome to be extraordinarily dynamic with changes in structure associated with particular gene sets and particular physiological and pathological processes. The same methods will allow us to associate topoisomerase activity with changes in DNA structure and gene activity. These studies will likely shed new light on how chemotherapeutic agents that alter DNA conformation and topology may be improved to increase efficacy and improve specificity. Toposiomerases are enzymes that control the distribution and level of supercoiling and torsional stress in DNA fibers. We have begun to analyze where type I and type II topoisomerase act across the genome to assess their relationship to gene activity and DNA structure. We are using ChIP to map sites of topoisomerase 1 binding across the genome to overlay the sites of top 1 binding on top maps of supercoiling and alternative DNA structures. We have discovered that there is tight coupling between RNA polymerase recruitment and topoisomerase action at the promoters of active genes. Moreover, topoisomerase 1 is used as a component of the transcription machinery independently of its enzymatic activity. In fact topoisomerase 1 activity is held in check until the topoiosmerase is recruited beyond 1 kilobase downstream of the transcription start site.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Kouzine, Fedor; Gupta, Ashutosh; Baranello, Laura et al. (2013) Transcription-dependent dynamic supercoiling is a short-range genomic force. Nat Struct Mol Biol 20:396-403