Modulation of Escherichia coli sister chromosome cohesion by topoisomerase IV

Abstract

A body of evidence supports the idea that newly replicated Escherichia coli chromosomes segregate progressively as replication progresses, with spatial separation of sister genetic loci occurring ∼15 min after their replication. We show that the time of this cohesion can be modulated by topoisomerase IV (TopoIV) activity. Impairment of TopoIV prevents segregation of newly replicated sister loci and bulk chromosome segregation, whereas modest increases in TopoIV decrease the cohesion time substantially. Therefore, we propose that precatenanes, which form as replication progresses by interwinding of newly replicated sister chromosomes, are responsible for E. coli sister chromosome cohesion. The heritable maintenance of the genetic material over generations requires not only that it be accurately replicated but that newly replicated chromosomes are faithfully transmitted to daughter cells at cell division. Defects in chromosome segregation are associated with genetic disease and cancer. In eukaryotes, newly replicated sister chromosomes remain associated and aligned until the onset of mitosis by dedicated cohesion mechanisms, using SMC (structural maintenance of chromosomes) proteins. Formation and dissolution of cohesion is under strict spatial and temporal control (for review, see Nasmyth and Haering 2005). Similarly, in the archaeon, Sulfolobus sulfataricus, sister cohesion seems to extend from the replicative to the post-replicative phase of the cell cycle (Robinson et al. 2007). In contrast, a growing body of evidence supports the view that newly replicated loci in bacteria segregate progressively as replication proceeds, with a period of sister cohesion (the time between locus replication and separation of the two sister loci) that is substantially less than S phase (Viollier et al. 2004; Neilsen et al. 2006;Reyes-Lamothe et al. 2008). Nevertheless, some work has led to the conclusion that extensive sister cohesion may hold sisters together for a large fraction of S phase (Sunako et al. 2001;Bates and Kleckner 2005).Topological entanglement of newly replicated sisters has been implicated in facilitating sister chromosome cohesion in bacteria, archaea, and eukaryotes, with precatenanes (interwound sister DNA duplexes present in a replicating chromosome), catenanes (interwound replicated sister duplexes), or hemicatenanes (duplexes inter- The right-hand (RH) interwinding of the two strands of a DNA duplex (linkage) has to be completely removed in order that newly replicated chromosomes can be segregated to daughter cells. Complete segregation of newly replicated Escherichia coli chromosomes requires the removal of ∼4.2 × 10 5 links in one generation time or less. RH precatenanes will arise when RH links in duplex DNA ahead of a progressing replication fork diffuse backward to behind the fork, thus interlinking the two newly replicated sisters by a redistribution of total linkage ( Fig. 1A; Champoux and Been 1980;Espeli and Marians 2004). In the absence of topoisomerase action, replication progression leads to a...

Independent Segregation of the Two Arms of theEscherichia coli oriRegion Requires neither RNA Synthesis nor MreB Dynamics

“…An image was taken every 10 min. lication initiation occurs efficiently under these conditions, most of the newly replicated loci have not segregated (47,59). By 70 min after replication initiation, the rifampin-treated and rifampin-free cultures showed essentially identical distributions of foci, with Ͼ80% of the cells containing two or more ori1 foci.…”

Species-specific supercoil dynamics of the bacterial nucleoid

“…Progress in understanding the structure/function mechanics of the bacterial chromosome greatly improved with development of techniques that allow real time visualization of bacterial nucleoids inside living cells and the ability to follow specific chromosomal loci using fluorescent labels (Kleckner et al 2014;Wang et al 2008). Nucleoids are self-adherent filaments stochastically organized locally into approximately 10-kb domains in growing cells (Cunha et al 2005;Espeli et al 2008;Hadizadeh Yazdi et al 2012;Postow et al 2004;Stein et al 2005;Wiggins et al 2010).…”

“Breaking Up Is Hard to Do”: The Formation and Resolution of Sister Chromatid Intertwines

“…However, there is direct evidence that some fork rotation does take place during elongation in E. coli and indirect evidence that fork rotation occurs during elongation in eukaryotic systems. In E. coli, cytological marking of DNA loci with fluorescent proteins has shown that a number of newly replicated loci appear "cohesed" for a short period following replication [36]. The time of cohesion following replication is directly related to topo IV dosage, with decreased dosage increasing the time of cohesion and increased dosage reducing it [36], arguing that the cohesion was maintained by DNA catenations formed by fork rotation and resolved by topo IV action.…”

“…In E. coli, cytological marking of DNA loci with fluorescent proteins has shown that a number of newly replicated loci appear "cohesed" for a short period following replication [36]. The time of cohesion following replication is directly related to topo IV dosage, with decreased dosage increasing the time of cohesion and increased dosage reducing it [36], arguing that the cohesion was maintained by DNA catenations formed by fork rotation and resolved by topo IV action. Therefore it seems reasonable to assume that a small amount of fork rotation and DNA catenation occurs during elongation to maintain transient cohesion behind the fork.…”

“…In vitro topo IV can support elongation rates that are comparable to those of gyrase [23]. However, the elongation rate of DNA replication in vivo drops to a third of wild type when DNA gyrase is specifically inhibited [35] whereas loss of topo IV activity has little influence on replication rates [36]. Therefore, in vivo, topo IV does not have an equivalent role to gyrase during elongation of DNA replication.…”