Processive Activity of Replicative DNA Polymerases in the Replisome of Live Eukaryotic Cells

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome. © 2020 Elsevier Inc.By establishing single-molecule methods in live budding yeast, Kapadia et al. measured the binding kinetics in the eukaryotic replisome. They show that the leading and lagging strand polymerases, Pol δ and Pol ε, are stably bound to the replisome. In contrast, Pol α primase performs few priming cycles before dissociating. © 2020 Elsevier Inc.