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Figure 11 Topoisomerase IV acts at the end of the cell cycle. A, the cell-killing assay;
B, TUNEL staining, as described in the text. |
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To prove this model, we developed assays to assess the location of both DNA gyrase and Topo IV on the nucleoid in vivo (Figure 11). Quinolone-induced, DNA gyrase-, or Topo IV-mediated cell killing as a function of the cell cycle was measured to determine when gyrase and Topo IV activity was manifest. An isogenic pair of strains was used where the GyrA subunit of DNA gyrase was either sensitive or resistant to the quinolone norfloxacin, and that also carried a temperature-sensitive dnaC mutation that allowed synchronization of cell growth by a triple temperature-shift procedure. Formation of quinolone-induced double-strand breaks correlates with the cytotoxicity of these drugs and has been shown to occur when the replication fork encounters the frozen topoisomerase-quinolone-DNA complex. When gyrase was sensitive to norfloxacin, cell killing was co-incident with the onset of replication, consistent with random positioning of gyrase on the DNA.
On the other hand, when gyrase was resistant to norfloxacin, cell killing occurred only late in the cell cycle, implying that Topo IV was restricted from access to the DNA until the end of the replication cycle. This interpretation was supported by the use of TUNEL to label all quinolone-mediated, SDS-induced, double-strand breaks on the nucleoid across the cell cycle. This technique should measure residence of any gyrase or Topo IV molecule on the DNA. The results were in complete agreement with the cell-killing experiments and showed that Topo IV activity was concentrated on the edge of the nucleoid proximal that was proximal to the septum.
We suggest that the temporal regulation affected by the sequestration of the Topo IV subunits from one another, coupled with the cell cycle-dependent release of ParC from the replication factory, is intended to focus Topo IV activity in rapidly growing cells to the correct pair of chromosomes in the cell -- those whose topological linkage must be eliminated in order for the cell to divide successfully. In cells that are growing slowly, the replication period for any particular chromosome is shorter than that of the complete cell cycle. Thus, the parental chromosome replicates, the daughters are partitioned, and the cells divide. Chromosome content does not exceed 2 per cell. Two conditions, therefore, obtain with respect to daughter chromosome decatenation: Topo IV bound anywhere on either of the daughter chromosomes can affect efficient decatenation, and thus uniformly dispersed Topo IV can effectively find the proper substrate, i.e., the chromosomes that have to be separated to allow subsequent cytokinesis.
Rapidly growing cells have more than one replicating chromosome and thus have multiple pairs of daughter chromosomes. Yet, successful cell division requires the decatenation of a particular pair of daughter chromosomes. Under conditions of rapid growth, we suggest that decatenation by uniformly dispersed Topo IV of the separating pair of chromosomes would be inefficient because by binding to all the other DNA in the cell, the enzyme is effectively occupied elsewhere. By temporally regulating Topo IV activity, the cell ensures that a bolus of Topo IV activity is delivered where it is needed -- to the chromosome pair that is clearing the division plane. As described in the next section, Topo IV activity is likely to be retained in the center of the cell because of an interaction between ParC and the septal ring protein FtsK.
