Sister ori1 and L2 genetic loci fail to segregate normally when TopoIV is impaired. (A–D) Snapshot images of cells, with ori1 labeled (red). (Blue) DAPI nucleoid staining. (E) Quantitation of the data from the experiments shown in A–D. Cells were classified into the four categories shown in the schematic. (Blue) Cells with one ori1 focus, and longer cells (>4 μm) with two closely spaced ori1 foci (d < 1/5 cell length). (Red) Shorter cells (<4 μm) with two closely spaced foci. (Green) Cells with well-separated foci (d ≥ 1/5 cell length) or two clusters of separated foci. (Purple) Cells with more than two foci or clusters of foci. The cells in the red group were distinguished from those in the blue group because we cannot be sure whether those in the red group have foci that are unable to separate normally or are on the way to normal separation. (F–H) Simultaneous labeling of ori1 (green) and L2 (red) genetic loci. Bars, 2 μm.

Sister replisomes migrate to separate cell halves in the absence of locus segregation. (A,B) Snapshot analysis of ori1 and replisome (Ssb-Ypet) in parE^ts strain at 30°C and 42°C (2 h). (C) Time-lapse analysis (5 min). Ten parE^ts cells were switched to 42°C (35 min) and then followed at 42°C by time lapse, and the mean positions of the ori1 foci (either a single focus or transient pair of closely spaced foci) and Ssb-Ypet foci are shown (bars show standard errors). (D) ori1 separation in parE^ts at 42°C (data from C) compared with ori1 separation in TopoIV⁺ cells grown at 42°C (not shown). The indicated time intervals are the time from replisome appearance to the last point at which a replisome was present. (E) The separation between sister replication forks after initiation from the data presented in C is compared with a similar experiment using wild-type cells grown at 37°C.

TopoIV impairment prevents sister nucleoid separation. (A) Schematic of topological processing during DNA replication. As a replication fork moves forward, the DNA ahead of the fork becomes overwound and the density of (+) RH linkage increases, forming (+) supercoils (sc) with a LH plectonemic superhelix ahead of the fork. The torsional stress so generated is relieved if rotation of the fork replisome occurs, thereby redistributing the RH linkage. This leads to formation of (+) RH precatenanes behind the fork. These are only good substrates for TopoIV if their density is low (“loose” precat), so their crossing angle can become optimal for TopoIV action (Stone et al. 2003). If precatenane-containing DNA becomes underwound ahead of the fork, generating (−) sc, then diffusion forward of RH precatenanes to remove (−) sc will be favored. It has also been proposed that accumulation of (−) sc ahead of a fork can lead to LH (−) precatenanes in newly replicated DNA, with these also being an in vivo substrate for TopoIV (see Schvartzman and Stasiak 2004). We believe this is unlikely to be a common occurrence, because it would exacerbate chromosome unlinking and be energetically unfavorable because ATP hydrolysis by gyrase ahead of a fork, generating (−) supercoiling, would need to be countered by ATP hydrolysis by TopoIV to remove LH precatenanes. Unreplicated DNA is shown as a thick black line and newly replicated DNA is shown as a gray line. Arrows indicate the direction of reaction. Thick arrows indicate efficient reactions and dashed arrows inefficient reactions. (B) Schematic of the E. coli genetic loci used. (C) Nucleoids in the indicated cells. Replication was blocked by TetR binding to tetO arrays at R3, ∼700 kb anti-clockwise of dif. White arrows indicate positions of nucleoid splitting and yellow arrows show positions of cytokinesis in a bright-field image. Nucleoids were stained with DAPI (0.5 μg/mL). Bars, 2 μm. (D) Flow cytometry (DNA content per cell) of wild-type and parE^ts cells grown at 30°C and 42°C. Rifampicin, which prevents replication initiation, and cephalexin, which blocks cytokinesis, were added at 0 min, and samples were taken at the indicated times.

Increased TopoIV decreases sister cohesion time. (A) Snapshot analysis in dnaC^ts TopoIV⁺ cells containing either an “empty” plasmid (pBAD24) or a plasmid with basal low-level expression of TopoIV (pWX35). The main plot shows the increase in cells containing two ori1 foci in a single experiment carried out over 120 min at 30°C from replication initiation. The inset shows three experiments carried out for a shorter time, with the mean values shown as thick lines and standard errors as vertical bars. (B) Time-lapse analysis of ori1 segregation in DnaC⁺ cells from replisome appearance at initiation.

Model for cellular organization of replication and chromosome segregation (see the text for details).