Replication restart in gyrB Escherichia coli mutants.
Gyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases. Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected. We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants. The helicase function of PriA is not essential. The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains. The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co-lethal in the presence of a DeltatopA mutation. Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate. We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA-dependent primosome assembly, directly on the in-activated replication forks, without the formation of a recombination intermediate.
Grompone
Gianfranco
G
Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, France.
Ehrlich
S Dusko
SD
Michel
Bénédicte
B
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D027081:DNA Gyrase / Q000235:genetics* / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D017930:Genes, Reporter; D009154:Mutation; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D013696:Temperature
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; C069996:PriB protein (N-terminal); C069995:PriC protein, E coli; D000251:Adenosine Triphosphatases; C506743:priA protein, E coli; D004265:DNA Helicases; D027081:DNA Gyrase