Polymerase exchange during Okazaki fragment synthesis observed in living cells |
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Authors: | Lia Giuseppe Michel Bénédicte Allemand Jean-François |
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Institution: | CNRS, Centre de Génétique Moléculaire, UPR3404, Gif-sur-Yvette F-91198, France. lia@cgm.cnrs-gif.fr |
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Abstract: | DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis. |
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