Activities of gyrase and topoisomerase IV on positively supercoiled DNA

Abstract

Although bacterial gyrase and topoisomerase IV have critical interactions with positively supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and topoisomerase IV to relax and cleave positively supercoiled DNA were analyzed. Gyrase removed positive supercoils ∼10-fold more rapidly and more processively than it introduced negative supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positive supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positively supercoiled substrates ∼3-fold faster than negatively supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positively supercoiled (compared with negatively supercoiled) DNA, whereas topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than topoisomerase IV to safely remove positive supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positive supercoils, rather than induction of negative supercoils.

Single-molecule imaging of DNA gyrase activity in livingEscherichia coli

“…To allow the replisome to maintain its incredibly high translocation rate, the two type II topoisomerases must relax up to 100 (+) supercoils per second for each fork (assuming a replisome translocation rate of 1000 bp/s, and DNA helical repeat of ~10bp) ( Figure 1B). In vitro, the catalytic cycle for both gyrase and topo IV has been measured at ~2 s, with each cycle removing 2 supercoils (17)(18)(19), suggesting that up to 100 enzymes would be required per fork to keep up with the replication rate in live bacteria. Early studies of chromosome fragmentation in E. coli cells using the gyrase targeting drug, oxolinic acid (20), suggested that gyrase may be clustered near the replication fork.…”

“…Bacillus anthracis gyrase suggests that gyrase 'bursting' activity might relax high levels of (+) supercoiling at faster rates (19). It remains to be established whether gyrase behaves processively or not in vivo, and whether its catalytic mode depends on the local supercoiling environment.…”

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts

“…The relaxation of both positive and negative supercoils is an essential process in all cells. In B. subtilis , relaxation of positive supercoils is accomplished by the activity of either DNA gyrase or Topo IV (Vos et al 2011;Postow, Crisona, et al 2001;Crisona et al 2000;Ashley et al 2017) . If the model of positive supercoil accumulation at head-on conflict regions is correct, then these enzymes should preferentially associate with a head-on conflict region.…”

“…Topo I relaxes negative supercoils produced by transcribing RNAPs. DNA Gyrase and Topo IV relax positive supercoils and have both been shown to be important for replication fork progression in vivo (Khodursky et al 2000;Crisona et al 2000;Peng and Marians 1993;Ashley et al 2017;Vos et al 2011) . Topo IV also plays critical roles in the separation of sister chromatids during segregation (Hiasa and Marians 1996;Zechiedrich and Cozzarelli 1995) .…”

Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoisomerase IIα

“…48–50 Consequently, is possible that the xanthone derivatives inhibit the activity of topoisomerase IIα in a bimodal fashion with the aromatic core of one molecule binding to the protein and the polyamine tail of another acting through a general effect on the DNA. Therefore, the importance of the linkage between the spermidine/spermine polyamine tails and the xanthone core (compound 6 ) in a single molecule was examined.…”