Eukaryotic Lagging Strand Synthesis is Distributive

DNA’s anti-parallel structure presents a topological challenge to the replisome, as the leading and lagging strands must be synthesized in opposite directions. It is widely believed that the lagging strand polymerases Pol δ and Pol α are stably tethered to the eukaryotic replisome to facilitate their recycling and to coordinate leading and lagging strand synthesis. To test this idea, we directly visualized the dynamics of Pol α/δ at active replication forks via single-molecule imaging in Xenopus nuclear extracts. Surprisingly, we find that neither Pol α nor Pol δ is stably tethered to the eukaryotic replisome. Instead, lagging strand synthesis occurs distributively and entails the recruitment of new molecules of Pol α and Pol δ to facilitate the synthesis of new Okazaki fragments. Our data reveal a highly dynamic mechanism of lagging strand synthesis and challenge the current textbook model of the eukaryotic replisome.