Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli

Abstract

The replication period of Escherichia coli cells grown in rich medium lasts longer than one generation. Initiation thus occurs in the 'mother-' or 'grandmother generation'. Sister origins in such cells were found to be colocalized for an entire generation or more, whereas sister origins in slow-growing cells were colocalized for about 0.1-0.2 generations. The role of origin inactivation (sequestration) by the SeqA protein in origin colocalization was studied by comparing sequestration-deficient mutants with wild-type cells. Cells with mutant, non-sequesterable origins showed wild-type colocalization of sister origins. In contrast, cells unable to sequester new origins due to loss of SeqA, showed aberrant localization of origins indicating a lack of organization of new origins. In these cells, aberrant replisome organization was also found. These results suggest that correct organization of sister origins and sister replisomes is dependent on the binding of SeqA protein to newly formed DNA at the replication forks, but independent of origin sequestration. In agreement, in vitro experiments indicate that SeqA is capable of pairing newly replicated DNA molecules.

Figure 1

Replication pattern of rapidly growing E. coli wild-type cells. Cells (yellow) with chromosomes (blue lines) and origins (black squares) are drawn schematically to show the number of replication forks and origins at different stages of the cell cycle. In this example, initiation of replication occurs at four origins at the same time as cell division (bottom). A young cell therefore contains four origins and six replication forks (upper left). As replication proceeds, the oldest pair of forks reach the terminus and the two sister chromosomes segregate. The cell then contains four origins and four replication forks (upper right). Initiation then occurs again at 4 origins and generates 8 new forks giving a total of 12 forks, as cell division approaches (bottom). Because there will be cell-to-cell variability, some cells will contain eight origins before they divide, whereas cells that divide before initiation of replication will contain only two origins (not shown). However, the majority of the cells in the culture will contain four origins.

Figure 2

Origin and replication fork localization in rapidly growing wild-type cells. Cells were grown in glu-CAA medium. (A) Fluorescent micrographs of SF72 cells in which the origin region was visualized using a GFP-LacI/lac operator system (left panel). The number of origin foci per cell was tallied (top right panel), and the number of origins per cell was determined by flow cytometry analysis (bottom right panel). (B) Immunofluorescence of BrdU-labelled MG1655 cells (left panel) and distribution of replication fork foci per cell (top right panel). The number of replication forks per cell (bottom right panel) was calculated from flow cytometry histograms. The cells with origin and replication fork foci that could not be determined were 5 and 14%, respectively, of the examined population of cells.

Figure 3

Origin and replication fork localization in oriCm3 and ΔseqA mutant cells. (A, C) Visualization of the origin region using the GFP-LacI/lac operator system in oriCm3 (A) and ΔseqA (C) cells (top panels). The number of origin foci per cell was counted in the oriCm3 mutant cells (A, bottom panel), but not in the ΔseqA mutant cells (C) (see Results). Immunofluorescence of BrdU-labelled oriCm3 (B) and ΔseqA (D) cells (top panels) and distribution of replication fork foci per cell (bottom panels). Of the oriCm3 and ΔseqA cells, 18 and 26%, respectively, had an undeterminable number of replication fork foci. Two percent of the oriCm3 cells examined had an undeterminable number of origin foci.

Figure 4

Cohesion of newly replicated origins by SeqA during bidirectional in vitro DNA replication. Supercoiled oriC plasmid (pBSoriC) was mixed with replication proteins and SeqA or SeqA mutant protein at 4°C. Replication was then performed at 30°C for 15 min, followed by restriction enzyme digestion with EcoRI and PstI (A). Native agarose (1.2%) gel electrophoresis of non-replicated pBSoriC (cut with EcoRI) (lane 1), replicated pBSoriC (supercoiled) (lane 2), replicated pBSoriC (cut with EcoRI and PstI) (lane 3), pBSoriC replicated in the presence of SeqA (lanes 4–6), SeqA2 (lanes 7–9) or SeqA4 (lanes 10–12) (cut with EcoRI and PstI), at 30, 120 and 360 nM (B).

Figure 5

Origin organization at four different growth rates. Cells (yellow) with chromosomes (blue lines) and origins (black squares) are drawn schematically to show the number of replication forks and origins at different stages of the cell cycles. Based on the observed distributions of origin foci per cell (Supplementary Table SI online), origin colocalization at these stages are indicated with green, filled circles. Each green circle represents a single copy of the origin. Two or four overlapping green circles represent foci containing two or four origins, respectively. (A) The replication and segregation period is confined within one generation time (T_d>C+D). Here, a newborn cell has one chromosome with one origin and one origin focus. After replication, the two new sister origins are colocalized for 10–20 min (Wang et al, 2005; Bates and Kleckner, 2005; Nielsen et al, 2006), and then move apart. The colocalization period may correspond in duration to the sequestration period. (B) The C+D period is longer than the generation time. Initiation of replication occurs at two origins and the two new sister origins on each mother chromosome are colocalized. The pairs of sister origins in each cell half separate right before or right after cell division, so that some of the newborn cells have one focus and some have two foci. Cells with three foci (see Supplementary Table SI) probably arise, because the separation of colocalized origins in the two cell halves occurs at somewhat different times. (C) The C+D period approximately equals two generations. Initiation of replication occurs at four origins around the same time as cell division. Pairs of sister origins remain colocalized throughout an entire round of replication and segregation, until they are initiated again and become four. In some of the cells, initiation occurs at four origins at the end of the ‘grandmother' generation and in some at two origins at the beginning of the ‘mother' generation. A newborn cell contains four origins that are either colocalized or have moved apart and become two separate sister pairs right before cell division. (D) The C+D period takes about two-and–a-half generations. Initiation of replication occurs at four origins in the ‘grandmother generation'. Four origins in each cell half are then colocalized for most of the remainder of that generation and separate into foci consisting of two sister origins either before or after cell division. Most newborn cells thus have four origins in two foci, while a few have four origins colocalized in one focus.