Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo.
We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.
Possoz
Christophe
C
Division of Molecular Genetics, Department of Biochemistry, University of Oxford, Oxford, UK.
Filipe
Sergio R
SR
Grainge
Ian
I
Sherratt
David J
DJ
D049109:Cell Proliferation; D002470:Cell Survival; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D018741:Replication Origin; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D013754:Tetracyclines / Q000378:metabolism
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013754:Tetracyclines; C016229:4-epianhydrotetracycline; D011938:Rec A Recombinases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli