Reconstituting human and SV40 replisomes with Okazaki fragment maturation reveals synchronized leading- and lagging-strand synthesis.

Eukaryotic lagging-strand synthesis requires iterative priming of Okazaki fragments (OFs) by Pol α, extension by Pol δ, and subsequent primer removal and ligation through Pol δ-mediated maturation of OFs (MOF). How the replisome coordinates these time-consuming lagging-strand processes while maintaining rapid and synchronized DNA synthesis remains unclear. Here, we reconstitute and compare the human and Simian Virus 40 (SV40) replisomes in the presence of MOF. In the human replisome, Pol α is recruited distributively to the CMG helicase for priming and transiently slows leading-strand synthesis by Pol ε, thereby directly coupling lagging-strand initiation to replisome progression. In contrast, Pol δ functions independently of CMG and remains highly distributive during both OF synthesis and MOF. Nevertheless, efficient recycling mechanisms of Pol δ maintain synchronization by prioritizing OF synthesis over MOF, resulting in prolonged accumulation of unligated OF intermediates without affecting leading-strand synthesis. In the SV40 replisome, where Pol δ directly interacts with the L-Tag helicase and synthesizes both DNA strands, leading-strand synthesis is similarly slowed by Pol α-dependent priming. However, MOF additionally slows leading-strand synthesis, revealing an additional layer of coordination required to maintain synchronization. These findings suggest a model in which the human replisome has evolved to simplify synchronization while maintaining replication speed and pave the way for a better understanding of its communication with other cellular processes.