Mechanisms of chiral discrimination by topoisomerase IV

Abstract

Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.DNA ͉ magnetic tweezers ͉ single molecule ͉ topology ͉ segregation T opological stress associated with excess supercoiling and knotted or interlinked DNA is resolved by an essential and conserved class of enzymes, the topoisomerases (1-4). Topoisomerases fall into two broad categories. Type I topoisomerases transiently cut a single strand of duplex DNA permitting them to relax overwound, positively supercoiled DNA [(ϩ)sc DNA], and underwound, negatively supercoiled DNA [(Ϫ)sc DNA]. Type II topoisomerases catalyze the ATP-dependent passage of one segment of double-stranded DNA (the transfer or T segment) through a second transiently cleaved segment of duplex DNA (the gate or G segment) (3). Type II topoisomerases can both relax and generate supercoils and, importantly, can unknot and unlink DNA. Cellular DNA topology is maintained through the concerted activity of these two classes of topoisomerases (5).Topo IV, a bacterial type II topoisomerase, is primarily responsible for unlinking replicated chromosomes in vivo (5-7). A summary of the in vivo roles and enzymatic cycle of Topo IV is presented in supporting information (SI) Fig. S1. In vitro, Topo IV relaxes (ϩ)sc DNA faster than (Ϫ)sc DNA. Paradoxically, the relaxation rate asymmetry is variable, ranging from a factor of Ϸ20 in ensemble measurements to a much larger value in single-molecule measurements (8-10). In contrast with Topo IV, the homologous yeast topoisomerase II (Topo II) relaxes positive and negative supercoils at the same rate (8). However, human topoisomerase II␣ preferential...

Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

“…Although both htopo II␣ and Topo IV exhibit chiral discrimination mediated by interactions between their CTDs and DNA writhe (17,20,21,24,25,28) (Fig. 4), our results suggest that the underlying mechanisms of discrimination differ.…”

“…Growing evidence suggests that CTD variations among Topo IIA enzymes also alter the topological specificity and activity of the otherwise similar core domain functions of prokaryotic and eukaryotic Topo IIA enzymes (17)(18)(19)(20)(21)(22)(23). For example, Escherichia coli topoisomerase IV (Topo IV) preferentially relaxes positive supercoils in a chiral discrimination process mediated by the CTDs (5,24,25). The molecular mechanisms underlying chiral discrimination by Topo IV have not been entirely elucidated, although it was shown to arise from chirality-dependent differences in enzymatic processivity, i.e.…”

“…The molecular mechanisms underlying chiral discrimination by Topo IV have not been entirely elucidated, although it was shown to arise from chirality-dependent differences in enzymatic processivity, i.e. the number of strand passages catalyzed per binding event (25). To date, an atomic structure of a eukaryotic Topo IIA CTD has not been solved, precluding a detailed structural comparison with prokaryotic CTDs.…”

The dynamic interplay between DNA topoisomerases and DNA topology

“…Both human Topo IIα and E coli Topo IV preferentially relax positive supercoils (Charvin et al 2003;Crisona et al 2000;McClendon et al 2005McClendon et al , 2008Neuman et al 2009;Seol et al 2013a;Stone et al 2003). The underlying mechanism for this chiral discrimination is embedded in differential interactions between positive and negative writhe and the C-terminal domains of the topoisomerases (Corbett et al 2005;McClendon et al 2008;Neuman et al 2009;Seol et al 2013a). However, the writhe-dependent chiral sensing differently affects the catalytic activities of the two enzymes as chiral discrimination by Topo IV results from chiralitydependent differences in processivity, whereas chiral discrimination by Topo IIα results from chirality-dependent differences in the relaxation rate (Neuman et al 2009;Seol et al 2013a) (Fig.…”

“…Single-molecule approaches complement ensemble approaches, but the former uniquely provide a means of distinguishing the effects of twist and writhe that are typically convolved in ensemble measurements. (Charvin et al 2003(Charvin et al , 2004(Charvin et al , 2005aNeuman et al 2009;Seol et al 2013a;Stone et al 2003). We can therefore distinguish twist (torsion) and writhe of DNA topology as the two main Beffectors^on DNA processing enzymes.…”