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

• Published in: Molecular cell 2020-10, Vol.80 (1), p.114-126.e8
• Main Authors: Kapadia, Nitin, El-Hajj, Ziad W., Zheng, Huan, Beattie, Thomas R., Yu, Angela, Reyes-Lamothe, Rodrigo
• Format: Article
• Language: English
• Subjects: budding yeast; DNA polymerase; DNA replication; live-cell imaging; replisome; Saccharomyces cerevisiae; single-molecule microscopy
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2020.08.014

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. [Display omitted] •Developed single-molecule methods to estimate binding kinetics in live yeast•Pol ε and Pol δ are stably associated with the replisome•Pol α is recycled about every minute•Ctf4 plays a minor role in retaining Pol α at the replisome 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.