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 PolA and PolD 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 PolA/D at active replication forks via single-molecule imaging in Xenopus nuclear extracts. Surprisingly, we find that neither PolA nor PolD is stably tethered to the eukaryotic replisome. Instead, lagging strand synthesis occurs distributively and entails the recruitment of new molecules of PolA and PolD 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.