This manipulation enables not only modification of DNA superhelicity to allow unwinding of the double helix, but allows the decatenation of circular DNAs, thereby enabling circular chromosomes or plasmids to be separated during cell division [1–3]. In Escherichia coli one of the best studied examples of a type IA Thiazovivin chemical structure topoisomerase (where the protein link is to the 5′ phosphate, in contrast to type IB topoisomerases where the protein link is to the 3′ phosphate) is DNA topoisomerase I, which is encoded by the topA gene. Topoisomerase I relaxes negative torsional stress and is required to ARRY-438162 research buy prevent the chromosomal DNA from becoming extensively

negatively supercoiled [4]. Topoisomerase 4EGI-1 molecular weight I requires an exposed single stranded region [4]. In E. coli the chromosomal DNA is normally slightly negatively supercoiled due to the activity of DNA gyrase, a type IIA topoisomerase, and extensive single stranded regions are not available for topoisomerase I to act on [3]. However, the unwinding of the double helix will result not only in single stranded regions but also in extensive changes in the local level of torsional stress.

For instance, the “”twin-domain”" model of transcription suggests that the elongating RNA polymerase complex (RNAP) causes accumulation of positive torsional stress in front of the transcription complex, whereas negative supercoils accumulate behind Celecoxib [5]. While the positive supercoils are relaxed by gyrase, the negative torsional stress leads to the formation of single stranded DNA, which is a hot-spot for relaxation by topoisomerase I [4]. In cells lacking the activity of topoisomerase I the chromosomal DNA becomes hypernegatively supercoiled, especially behind transcribing RNAP complexes. DNA gyrase will remove the positive torsional stress in front of RNAP, whereas the negative supercoils will persist if they cannot be relaxed by Topo I. This accumulation of negative supercoils has been thought to increase the probability that the newly generated transcript will hybridise with the

template strand, thereby forming an R-loop [6]. This idea was supported by results showing that R-loops are a substrate for topoisomerase I in vitro [4]. Furthermore, increased levels of RNase HI, encoded by the rnhA gene, have been shown to partially suppress the growth defect of ΔtopA cells, while the deletion of rnhA exacerbated the ΔtopA phenotype [7]. It was initially described that ΔtopA cells can grow without apparent ill effect [8]. However, it was later discovered that the ΔtopA mutant strains used had accumulated compensatory mutations in DNA gyrase and that ΔtopA strains without these suppressor mutations show a severe growth defect [9], an observation confirmed in later studies [7]. It is not clear why growth of cells lacking topoisomerase I is so severely impeded.