FtsK and Topo IV — Does the Cell Contain a Decatenosome?

[Enlarge Image] Figure 12 The C-terminal domain of FtsK stimulates decatenation catalyzed by topoisomerase IV but not by DNA gyrase.

The principal role of Topo IV in the cell is to effect daughter chromosome decatenation. This reaction can be modeled in vitro with the use of multiply-linked DNA dimers as a substrate. These molecules are purified from DNA replication reactions where DNA ligase has been omitted and thus the product DNA is form II. Therefore, when analyzed by agarose gel electrophoresis, the form II:form II DNA dimers appear as a ladder of bands with a certain distribution, with each step in the ladder representing 1 intermolecular linkage between the 2 daughter DNAs (Figure 12). The reactions are quantitated by determining the total linkages present at 0 time and after the 5 min incubation, and then deriving a rate of decatenation (in fmol linkages decatenated/min). The presence of FtsK_C in this decatenation assay stimulated Topo IV activity 2.5-fold; whereas it inhibited DNA gyrase activity by 25 percent. Thus, although the observed stimulation of Topo IV activity could be indirect, the lack of a similar stimulation of gyrase activity argues strongly that there is a functional interaction between Topo IV and FtsK_C.

	[Enlarge Image] Figure 13 A model for the temporal and spatial regulation of topoisomerase IV activity in the cell. The model is described in the text.

We have constructed a working framework to explain the observations outlined in the previous sections. The cell cycle of E. coli growing rapidly in rich medium is represented in Figure 13 from the standpoint of residence of proteins at the replication factory and the septal ring. Four successive steps are represented (a to d). We presume that these steps are not of equal length in time. We posit that steps c and d are short compared with steps a and b (about 40 min together). Figure 11a represents a newborn cell that has initiated DNA replication. There is one ParC/replication factory focus at mid-cell. ParE can be found at mid-cell in a small fraction of cells, possibly because of its association with an unknown factor. Because the ParC is effectively trapped in the replication factory by its interactions with / and , even though some ParE and ParC are spatially co-resident, there is no Topo IV activity.

Figure 13b represents the situation as DNA replication nears termination. The duplicated origin regions have moved into distinct halves of the cell. The terminus region for replication is co-localized with the parental factory. At this point, the second round of initiation leads to the generation of 2 additional ParC/replication factory foci that are located at the quarters of the cell (not shown in the cartoon). The cell is preparing to divide; the septal ring has formed; FtsK has been recruited; and the ring is beginning to constrict. The concentration of ParE at mid-cell is increasing, but there is still no Topo IV activity. In Figure 13c, replication is in the terminal stage, and the parental factory has disassembled. The daughter chromosomes are still catenated, and the septal ring has constricted significantly. Free ParC, possibly in complex with /, is captured by FtsK_C at the septal ring and can interact with ParE.

Once formed, the active Topo IV presumably remains associated with the septal ring. Subsequent decatenation requires that Topo IV interact with the daughter chromosomes. We currently envision this occurring as a result of the XerCD-FtsK-Topo IV axis of protein-protein interactions physically directing Topo IV to the dif region. Decatenation now occurs as the septal ring is closing (Figure 13d). The fate of Topo IV at this point is unclear. We assume that following either decatenation or cytokinesis, the Topo IV heterotetramer at this septal location is disrupted. Much of our current research is focused on obtaining additional evidence for this model.