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Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells

Dynamic Replication In all organisms, DNA replication involves a multiprotein complex called the replisome. Active Escherichia coli replisomes contain three copies of DNA polymerase III (Pol III). Lia et al. (p. 328, published online 22 December) used single-molecule spectroscopy to probe the dynamics of single proteins during E. coli replication in vivo. The results confirmed the presence of three Pol III molecules in the active replisome, with regular exchange of one of these Pol III's with one from the pool. Coordination with single-stranded DNA content suggests the Pol III that performs lagging-strand synthesis is exchanged so that a new Pol III is used for the synthesis of each Okazaki fragment. Single-molecule microscopy suggests that a new core polymerase is used to synthesize each Okazaki fragment. 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.