In prokaryotes, factors that package DNA, such as HU proteins, may control supercoiling by binding to DNA and trapping the free energy of supercoiling as writhe and subsequently releasing it through controlled dissociation [ 3 and 4]. Similarly in eukaryotes the regulated release of terminal DNA from a nucleosome, mediated by the acetylation of core histone tails, could release constrained writhe for conversion into negative supercoiling. Although in

vitro studies support this concept [ 5] its operation in vivo is elusive [ 6]. In prokaryotes and eukaryotes all activities buy I-BET-762 that require DNA to be unwound (and rewound) are potent generators of supercoiling. The classic example is the ‘twin supercoiled domain’ model where elongating RNA polymerase, in unwinding the DNA, generates positive supercoiling ahead and, in rewinding the DNA, generates negative supercoiling in its wake [7 and 8] (Figure 1). The levels of supercoiling produced in this process are prodigious, amounting to a positive and a negative

supercoil for every 10 bp transcribed. Apitolisib molecular weight Consequently the role of topoisomerases in releasing torsional stress is crucial if the template is to be maintained in a transcriptionally competent state. Genes that are negatively supercoiled are generally more efficiently transcribed [9 and 10] but topoisomerase inhibition studies [11, 12•, 13 and 14] indicate that the accumulation of excessive positive or negative supercoiling will repress transcription. Therefore, there must be a regulated balance in the localised levels of supercoiling through the concerted actions of polymerases [15]

and topoisomerases [16 and 17]. When an activity supercoils Clomifene DNA the torque generated is transmitted along the molecule. If the ends of the molecule are not fixed (or at least hindered), the supercoiling will dissipate via the unhindered rotation of the helix. Therefore for supercoiling to have a structural or functional influence on DNA or chromatin it must operate within a constrained environment where the energy is at least transiently trapped or restricted. For this reason it is anticipated that genomes need to be organised into supercoiling domains with barriers that prevent the spread of topological stress. In prokaryotes the Escherichia coli genome has a hierarchical organisation based on large structural macrodomains [ 3] with the Ter domain being subdivided into smaller, 35 kb domains via MatS/MatP interactions [ 18]. This organisation establishes a dynamic structural architecture enabling packaging without interfering with transcription or replication. The genome is also separately organised into about 500 independent ∼10 kb supercoiling domains with demarcating barriers stochastically distributed and dynamically maintained [ 19 and 20].