The Replisomes Remain Spatially Proximal throughout the Cell Cycle in Bacteria.

The positioning of the DNA replication machinery (replisome) has been the subject of several studies. Two conflicting models for replisome localization have been proposed: In the Factory Model, sister replisomes remain spatially co-localized as the replicating DNA is translocated through a stationary replication factory. In the Track Model, sister replisomes translocate independently along a stationary DNA track and the replisomes are spatially separated for the majority of the cell cycle. Here, we used time-lapse imaging to observe and quantify the position of fluorescently labeled processivity-clamp (DnaN) complexes throughout the cell cycle in two highly-divergent bacterial model organisms: Bacillus subtilis and Escherichia coli. Because DnaN is a core component of the replication machinery, its localization patterns should be an appropriate proxy for replisome positioning in general. We present automated statistical analysis of DnaN positioning in large populations, which is essential due to the high degree of cell-to-cell variation. We find that both bacteria show remarkably similar DnaN positioning, where any potential separation of the two replication forks remains below the diffraction limit throughout the majority of the replication cycle. Additionally, the localization pattern of several other core replisome components is consistent with that of DnaN. These data altogether indicate that the two replication forks remain spatially co-localized and mostly function in close proximity throughout the replication cycle. The conservation of the observed localization patterns in these highly divergent species suggests that the subcellular positioning of the replisome is a functionally critical feature of DNA replication.