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- Experimental procedures
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The cellular function of Escherichia coli topoisomerase III remains elusive. We show that rescue of temperature-sensitive mutants in parE and parC (encoding the subunits of the chromosomal decatenase topoisomerase IV) at restrictive temperatures by high-copy suppressors is strictly dependent on topB (encoding topoisomerase III). Double mutants of parEΔtopB and parCΔtopB were barely viable, grew slowly, and were defective in chromosome segregation at permissive temperatures. The topB mutant phenotype did not result from accumulation of toxic recombination intermediates, because it was not relieved by mutations in either recQ or recA. In addition, in an otherwise wild-type genetic background, ΔtopB cells treated with the type II topoisomerase inhibitor novobiocin displayed aberrant chromosome segregation. This novobiocin sensitivity was attributable to an increased demand for topoisomerase IV and is unlikely to define a new role for topoisomerase III; therefore, these results suggest that topoisomerase III participates in orderly and efficient chromosome segregation in E. coli.
- Top of page
- Experimental procedures
- Supporting Information
Topoisomerases are essential enzymes that regulate and maintain the topological state of chromosomal DNA by making a transient DNA break, passing a DNA segment through that break, and subsequently resealing the break (Wigley, 1995; Champoux, 2001; Bates and Maxwell, 2005). Type I topoisomerases cleave and pass one strand of DNA, whereas type II enzymes cleave and pass both strands of DNA (Wigley, 1995; Champoux, 2001; Bates and Maxwell, 2005).
Topoisomerases are essential for managing DNA topology during all macromolecular transactions on chromosomal DNA. In particular, their activities ensure the removal of excess positive topological linkages that accumulate during DNA replication because of the unwinding of the parental DNA strands. Either a type I or type II enzyme can be effective at this task. Once replication is completed, any remaining linkages between the two parental strands are manifested as catenanes between the two daughter chromosomes. If the replicated strands are completely sealed, then only a type II enzyme can unlink the daughter molecules (Sundin and Varshavsky, 1980; 1981; Wang, 1991). Champoux and Been (Champoux & Been, 1980) noted that the excess positive linkages that accumulate ahead of the replication fork can diffuse behind the replisome to form windings of the partially replicated daughter duplexes about each other that have come to be called precatenanes (because once replication is completed they convert to catenanes). We demonstrated that precatenanes do form during replication of a circular DNA in vitro (Peter et al., 1998). Precatenanes can clearly be resolved by type II topoisomerases, however, we have suggested that a type I topoisomerase operating at or near the replication fork where single-stranded gaps are available could also unlink them (Nurse et al., 2003).
Escherichia coli harbours four DNA topoisomerases: two type I enzymes [topoisomerase (Topo) I and III] and two type II enzymes (DNA gyrase and Topo IV) (Bates and Maxwell, 2005). Extensive studies and the properties of these enzymes have implicated some of them in clearly defined roles in the cell. DNA gyrase (encoded by gyrA and gyrB) (Gellert et al., 1976a; Mizuuchi et al., 1978) is required for preserving the global negative supercoiled state of the chromosome (Champoux, 2001), which it does primarily via the conversion of positive supercoils generated by DNA replication and transcription directly to negative supercoils (Levine et al., 1998; Wang, 2002). Topo I (encoded by topA) is important for preventing the accumulation of excess negative supercoils by specifically relaxing negatively supercoiled DNA (Wang, 1991), thus joining with DNA gyrase to homeostatically regulate the global level of chromosomal supercoiling (Menzel and Gellert, 1983).
Topo IV is required for the complete topological separation of the linked daughter chromosomes generated by DNA replication and is essential for viability (Kato et al., 1990; Adams et al., 1992; Peng and Marians, 1993). Topo IV consists of a heterotetramer of ParE and ParC dimers (Kato et al., 1992; Peng and Marians, 1993). Temperature-sensitive mutants of parE and parC manifest a partitioning defect (par) (Kato et al., 1989; 1990) where cell division is arrested, leading to extreme filamentation coincident with the accumulation of one or two large masses of unsegregated, replicating DNA at the cell centre.
More is known about the activities of Topo III in vitro than its role in vivo. This enzyme binds tightly to single-stranded DNA and does not appear to have the ability to locally unwind duplex DNA to create a binding site for catalysis. Thus, it is a potent decatenase on DNA rings that have small gaps, but is very poor at relaxing supercoils (Srivenugopal et al., 1984; DiGate and Marians, 1988), suggesting that it is unlikely to contribute to the maintenance of global supercoiling. On the other hand, Topo III can support replication fork progression in the absence of either DNA gyrase or Topo IV, and generate completely unlinked daughter chromosomes (Hiasa and Marians, 1994). The ability of the enzyme to remove precatenanes is thought to contribute to these activities (Nurse et al., 2003).
Mutants of topB (encoding Topo III) are viable and do not display obvious chromosome abnormalities or growth defects, thus it is not clear if Topo III is capable of resolving catenanes in vivo, but these mutants do show an increase in deletions between short homologous regions and an increase in frameshift mutations, suggesting that cells lacking topB have either an increased incidence of recombination at short DNA repeats or DNA replication mispairing slippage (Yi et al., 1988; Schofield et al., 1992; Uematsu et al., 1997). In addition, topB mutations are synthetically lethal with topA (Zhu et al., 2001) and parE mutations (Lopez et al., 2005) and it has been reported that deletion of recA rescues the synthetic lethality in both cases, suggesting a role for topB in the resolution of recombination intermediates (Zhu et al., 2001; Lopez et al., 2005). This observation is consistent with the known role of Topo III in eukaryotes, where it has been shown to associate with various RecQ family helicases in resolving toxic, unscheduled recombination intermediates that can occur, for example, at stalled replication forks (Oakley and Hickson, 2002). Failure to resolve such intermediates can lead to chromosome breakage, chromosome rearrangements, and faulty chromosome segregation.
Functional cooperation between the E. coli enzymes has been demonstrated in vitro as well: Topo III and RecQ can catenate covalently closed, double-stranded DNA rings (Harmon et al., 1999) and catalyse the resolution of stalled, converging replication forks (Suski and Marians, 2008). Collectively, these data point to a role for Topo III in vivo of either clearing toxic recombination and replication intermediates or unlinking sister chromosomes during replication, possibly with the assistance of its cognate helicase RecQ.
To understand how Topo IV could be regulated and to identify other proteins involved in resolving topologically linked sister chromosomes, we identified high-copy suppressors of the temperature-sensitive (ts) alleles parE10 and parC1215. Overproduction of several replisome components: the γ and τ subunits of the DNA polymerase III holoenzyme (both encoded by dnaX) and the β processivity clamp (encoded by dnaN), as well as an integral membrane protein encoded by setB, and Topo III could suppress the Topo IV mutant par and growth defects at non-permissive temperatures (Levine and Marians, 1998; Nurse et al., 2003; Espeli et al., 2003b).
As a potent decatenase (DiGate and Marians, 1988), it was reasonable to speculate that Topo III, when overproduced, could suppress parE and parC ts mutations at the restrictive temperature by decatenation of linked sister chromosomes. However, the question remained: Does Topo III participate in the removal of catenanes and/or precatenanes under physiological conditions?
Suppressors of the parE10 and parC1215 mutations are likely to either play a role in chromosome segregation or influence Topo IV activity. Here we have investigated the role of these suppressors. We find that topB is required for the suppressors to promote growth of parE and parC ts mutant strains at restrictive temperatures, suggesting that the mechanism of suppression could be potentiation of Topo III activity. Combinations of ΔtopB with the Topo IV ts mutations were synthetically lethal at permissive temperatures and these double mutant strains exhibited chromosome segregation defects. Contrary to previously reported findings (Lopez et al., 2005), neither recQ nor recA were required for the parE10ΔtopB synthetic lethality, because mutations in recQ and recA did not rescue the parE10ΔtopB double mutant strain. Furthermore, in the absence of these recombination proteins, the high-copy suppressor γ [dnaX(γ)] is still unable to rescue the parE10ΔtopB double mutant strain at 30°C, arguing that the observed phenotype was not the result of the accumulation of toxic recombination intermediates. However, we discovered that mutants of topB are hypersensitive to the type II topoisomerase inhibitor novobiocin: when exposed to novobiocin these cells are less viable and have severe chromosome segregation defects. These data suggest that Topo III plays a role in orderly and efficient chromosome segregation.