Independent Positioning and Action of Escherichia coli Replisomes in Live Cells

Summary 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.


Construction of chromosomal encoded fluorescent repressors
Copies of the chimeric genes tetR-mcerulean and lacI-mcherry were integrated into the chromosome in order to avoid the use of plasmids, which could potentially alter the localization of the replisome.
For tetR-mcerulean, the gene mcerulean was amplified using the primers: 5'-aaggagctcggctggctccgctgctgtgagcaagggcgaggag-3' (forward) and 5'-taggctcccgggttacttgtacagctcgtcc-3'(reverse), resulting in a fragment containing the gene preceded by the sequence of a 6aa flexible linker. It was then cloned in pUC18 between SacI and BamHI sites along with a kanamycin resistance cassette obtained using the primers described above. The resulting plasmid was pROD7. The gene tetR was then amplified using the forward and reverse primers 5'-cttgaattcgtctagattagataaaagtaa-3'and 5'-cttgagctcgaaatgtcagacccactttcacatttaa-3' respectively, and cloned in pROD7 between EcoRI and SacI sites resulting in pROD14, which has an in-frame fusion regulated by a lac promoter. The promoter, gene fusion and resistance cassette were integrated in the chromosome by replacing the gene galK through λ-Red recombination, using the primers 5'-gtttgcgcgcagtcagcgatatccattttcgcgaatccggagtgtaagaacgcccaatacgcaaaccg-3'and 5'-cggctgaccatcgggtgccagtgcgggagtttcgttcagcactgtcctgccttatgaatatcctccttag-3'.
In the case of lacI-mcherry, a similar strategy to that of tetR-mcerulean was used, using the same primers to amplify the fluorescent protein, and the gene was cloned into pUC18 along with a resistance cassette for chloramphenicol (template pKD3, using the same primers as for kanamycin resistance), this plasmid was named pROD22. lacI was amplified using the primers: 5'-cttgaattcggtgaatgtgaaaccagtaacgttat-3'and 5'ttagagctcgaacccagctgcattaatgaatc-3', and cloned in pROD22 between EcoRI and SacI sites, resulting in pROD25. The fusion was inserted in the chromosome by replacing leuB using the primers: 5'-aaagagttgcaacgcaaagctcaacacaacgaaaacaacaaggaaaccgtcgcccaatacgcaaaccg-3' and 5'-gtcgaacaatttttcgtataacgtcttagccatgattacaccccttctgccttatgaatatcctccttag -3'.

Microscopy
For snapshot analysis, cultures were grown overnight in M9 with glycerol as carbon source and subcultured once in the same medium. Cells were obtained from cultures in the early logarithmic phase (A 600 0.1 and 0.2), concentrated, resuspended in PBS and laid on a 1 % agarose pad on a slide. Nucleoids were visualized by staining them with 4',6-Diamidino-2-phenylindole (DAPI) (final concentration of 1 µg/ml). For timelapse, cells were treated as before but were instead laid on an M9-glycerol 1% agarose pad. Slides were incubated for 30 minutes in the microscope incubator chamber at the temperature used in the respective experiment. Cells were visualized with a 100x objective on a Nikon Eclipse TE2000-U microscope, equipped with a Photometrics Cool-SNAP HQ CCD camera. Images were taken, analyzed and processed by Metamorph 6.2.

Flow cytometry
Cells were fixed with 70% v/v ethanol. Samples were washed twice with PBS and stained with Syto-16 (0.1 µg/ml). 100 000 events recorded in a Becton Dickinson FACScalibur machine using FL1-H. Datsenko, K.A., and Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U S A 97, 6640-6645. Figure S1. Growth and cell cycle parameters for replisome-fluorescent protein fusions Fusions to Ssb, DnaQ, HolC, DnaX, HolA, HolD and DnaE not only had generation times close to the wild type strain AB1157, but showed wild type flow cytometry profiles. Furthermore, in microscopy snapshots of steady state cells, the cell length profiles and relative proportions of 1 ori1 to 2 ori foci cells (when ori1 was labeled) was in the same range as for wild-type. This relative proportion gives a good indication of cell cycle parameters. For example, in Fig. 3D, 29% of ori1 DnaQ-YPet cells had a single ori1 focus as compared to 23% in Wang et al. (2005), while 24.7% of Ssb-YPet cells had 1 ori1 and 28.5% of HolC-YPet cells had 1 ori1 focus (not shown). Although viable, fluorescent DnaB fusions were not analyzed extensively because they grew poorly sometimes and then had less normal cell cycle parameters. Fusions to DnaG and DnaN were not viable. Figure S2.

Supplemental References
A. DnaQ and Ssb focus frequency in dnaA ts and dnaC ts strains, with corresponding flow cytometry analysis after runout at 42 o C (see Fig. 2). B. Correlation between ori1 and Ssb positioning at the time of Ssb focus appearance.
See Fig. 2B. Figure S3. Ssb trajectories for 12/50 timelapse series See Fig. 3. Examination shows that sister replisomes are each mobile with respect to midcell. An analysis of sister replisome mobility is given in Fig. 6 and associated text.

Figure S4. Ssb-L3-R3 trajectories
A map of the loci used is shown (top). See Figure 6A. Examination of sister foci with respect to midcell provides insight into the relative mobility of sister replisomes.