Independent positioning and action of Escherichia coli replisomes in live cells

Abstract

A prevalent view of DNA replication has been that it is carried out in fixed "replication factories." By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.

Figure 1

Replisome Localization (A) The cellular distribution of DnaQ, HolC, and Ssb foci. The distribution of cellular focus positions is shown for cells with one focus (blue) or two foci (green and red). The distance to the closest pole and side was determined for one focus cell. For cells with two foci, the most polar focus was always considered as focus 1 (green). The blue and green focus quartile positions are shown expanded to the whole cell and half cell, respectively, in gray. When two foci are present, their relative positions are conserved. (B) Number of replisome foci as a function of cell length. A cell population was divided into groups based on cell length and the proportion of cells with 0, 1, 2, and >2 DnaQ foci is presented for each group. The % cells with/without foci for the assayed replisome proteins is shown. HolA, C, and D are clamp loader components; DnaE is the replicative polymerase, PolII; DnaX is the replisome organizer/clamp loader. (C) Colocalization of Ssb and DnaQ. For a subpopulation of cells containing Ssb-CFP and DnaQ-YPet, the proportions of cells in the indicated categories are shown. The probability of a Ssb focus colocalizing with DnaQ was 91%.

Figure 2

Replisome Foci Appear at oriC on Replication Initiation (A) Initiation and focus appearance. The proportion of cells with/without Ssb foci is shown for cells carrying the dnaC2 allele when incubated at 42°C for one generation and 5 and 10 min after transfer to 30°C. For flow cytometry analysis, cultures were incubated for 120 min at 42°C after the indicated treatment to allow completion of DNA replication. (B) Timelapse (5 min intervals) analysis of wild-type (left) and ΔmukB cells (right). At the time of appearance of DnaQ foci, the correlation between DnaQ position at replication initiation and ori1 position was recorded. In ΔmukB cells, ori1 is aberrantly positioned close to the poles, immediately prior to initiation. ΔmukB cells were grown at 22°C and wild-type cells at 37°C. Foci are shown for cells sorted by length. Note that ΔmukB cells are often delayed in cell division and reinitiate replication prior to division. (C) Colocalization of replisome and stalled fork. Cells carrying a tetO ori1 array were induced to overexpress TetR-CFP to block replication at ori1. Localization of DnaQ was analyzed in long cells with one ori focus to ensure that they were blocked. The probability of colocalization of DnaQ and an ori1 focus was 81%. The presence of >2 ori foci in the cell shown is a consequence of new initiations occurring during the period of the block. Bar, 1 μm.

Figure 3

Replisomes and the Cell Cycle (A) The localization of Ssb was followed in 50 cells every 5 min for 60 min. Timelapse series were made according to the time after the appearance or the time before the disappearance of Ssb foci and are shown as two overlapping series. Cells having one focus are represented as blue dots, while cells with two foci are shown in green (first) and red (second). A set of images 10 min apart is shown for two representative cells. Bar, 1 μm. (B) Statistics of appearance, disappearance, and movement apart of Ssb foci at replication initiation and termination. (C) Snapshot analysis of DnaQ with respect to ori1 (left panel) and ter3 (right panel). A steady-state cell population was separated into classes according to cell length. The proportion of cells with one or two ori1 foci (gray or blue areas, respectively) and with zero, one, or twp DnaQ foci (blue, red, or green lines, respectively) is represented for each of the classes (left panel). A comparable analysis was done with DnaQ and ter3 (right panel).

Figure 4

Sister Cohesion The time of separation of sister ori1 foci (left panel) or ter3 foci (right panel), with respect to time of Ssb appearance or disappearance, respectively, is plotted. In the right panel ter3 separation is related to the last time at which Ssb is present.

Figure 5

Replisome Position Is Highly Dynamic (A) Movement of Ssb foci in 5 min intervals. Timelapse series for two cells show the position of Ssb foci on the long axis of the cell through time. In cells with one focus this is represented by a blue dot, and the second, third, and fourth foci are represented with red, light blue, and orange, respectively. (B) Movement of Ssb foci in 30 s intervals. The position of Ssb foci with respect to the cell long and short axes is shown. (C) Distribution of step sizes (long axis) in 3 s timelapse.

Figure 6

Relative Movement of Replisome and Genetic Loci (A) The position of Ssb relative to loci L3 and R3 was followed at intervals of 5 min in 21 timelapse series (also see Figure S4). (B) Mean focus positions are graphed with standard errors of the mean (SEMs) indicated by bars. The shaded areas emphasize the position of the loci relative to the replisome in the period before L3-R3 replication. (C) Net movements of Ssb with respect to L3-R3 from first appearance of the replisome to 35 min later. SD = standard deviation. (D) Relative movement of Ssb with respect to ori1 and initiation. (E) Relative movement of Ssb and ori1 in three cells.

Figure 7

The Replisome Is More Mobile than L3-R3 Genetic Loci (A) Dynamics of Ssb plotted as MSD against time. Left panel is linear plot and right panel is log plots. The broken line shows the curve expected for free diffusion (slope 1). Error bars represent SEM. (B) Relative movement of Ssb, L3, and R3 in long and short cell axes. Log plots only are shown. Linear plot data were used to compute apparent diffusion coefficients, Dapp. Error bars represent SEM. (C) Cartoon of a cell is shown in which ovoids of different colors represent the average distance traveled by the loci L3, R3, or Ssb in 5 s, 10 s, 15 s, and 25 s (inner to outer layers). Half of the length or width of the ovoid represents the average distance traveled in the long axis or short axis in the indicated times.