BPS2026 – This is not a trombone: Revisiting the textbook model of eukaryotic DNA replication

Organisms with double-stranded DNA genomes universally perform continuous leading strand synthesis and discontinuous lagging strand synthesis. In the eukaryotic replisome, polymerase epsilon (PolE) synthesizes the leading strand continuously, while polymerase alpha (PolA) and polymerase delta (PolD) synthesize the lagging strand discontinuously. DNA’s anti-parallel structure presents a topological challenge to the replisome, as the leading and lagging strands must be synthesized in opposite directions. Early studies of bacteriophage replication revealed that both the DNA primase and the lagging strand DNA polymerase are stably tethered to the bacteriophage replisome, enabling it to processively synthesize many consecutive Okazaki fragments. To rationalize these observations, it was proposed that the lagging strand DNA template loops to facilitate the coordinated synthesis of leading and lagging strands by a single stable enzyme complex. This was coined as the “trombone” model, referring to the trombone-like loop formed by the lagging strand template. While originally proposed for bacteriophage replication, the trombone model is accepted as the textbook model of eukaryotic DNA replication. 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.