Kapadia NEl-Hajj Z.WZheng HBeattie T.RANGELA YU-CHEN LINReyes-Lamothe R.2022-11-162022-11-16202010972765https://www.scopus.com/inward/record.uri?eid=2-s2.0-85091677247&doi=10.1016%2fj.molcel.2020.08.014&partnerID=40&md5=c3b3c3b0ea6b0f154fd6665366e58bf8https://scholars.lib.ntu.edu.tw/handle/123456789/625597DNA 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.budding yeast; DNA polymerase; DNA replication; live-cell imaging; replisome; Saccharomyces cerevisiae; single-molecule microscopyDNA polymerase; nuclear protein; Okazaki fragment; rfa1 protein; rpa protein; unclassified drug; DNA directed DNA polymerase; DNA synthesome; multienzyme complex; nuclear protein; Saccharomyces cerevisiae protein; Article; binding kinetics; DNA determination; DNA replication; DNA synthesis; eukaryotic cell; fluorescence microscopy; fluorescence recovery after photobleaching; nonhuman; protein structure; replisome; retention time; Saccharomyces cerevisiae; biological model; cell nucleus; DNA replication; eukaryotic cell; kinetics; metabolism; protein subunit; reproducibility; Saccharomyces cerevisiae; single molecule imaging; time factor; Cell Nucleus; DNA Replication; DNA-Directed DNA Polymerase; Eukaryotic Cells; Kinetics; Models, Biological; Multienzyme Complexes; Nuclear Proteins; Protein Subunits; Reproducibility of Results; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Single Molecule Imaging; Time Factorsjournal article10.1016/j.molcel.2020.08.014329160942-s2.0-85091677247