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Abstract
The Escherichia coli SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, E. coli cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2–6 minutes and resulted in SeqA foci localized at each of the cell’s quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.
Highlights
DNA replication in the bacterium Escherichia coli is initiated at the replication origin, oriC, and proceeds bidirectionally towards the terminus, Ter [1]
We have investigated segregation patterns of newly replicated DNA through the cell cycle in rapidly growing E. coli cells using fluorescently labeled SeqA protein (SeqA-YFP)
Flow cytometry analysis showed that initiation of replication occurred simultaneously at all origins present in the cell (Text S1 and Figure S1). This indicated that origin sequestration was not compromised by the YFP-tagging of the SeqA protein
Summary
DNA replication in the bacterium Escherichia coli is initiated at the replication origin, oriC, and proceeds bidirectionally towards the terminus, Ter [1]. In slowly growing E. coli cells initiation of replication occurs at one origin and the circular chromosome is organized into a dynamic helical ellipsoid [2] with the left and right replichores in separate cell halves before replication [3,4]. The origin is situated at midcell and the two newly replicated origins stay colocalized there for about 20 minutes [5,6,7,8] Following this period of colocalization specific translocation of the sister origins occurs, possibly via the centromere-like migS site [9] and/or other unknown mechanisms, to each of the cell halves. Transient colocalization of sister chromosomes [6,11,12,13,14,15] and proper chromosome organization by the MukBEF complex [16,17,18] has been shown to be important to ensure proper chromosome segregation
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