CryoEM insights into RNA primer synthesis by the human primosome

Eukaryotic DNA replication depends on the primosome – a complex of DNA polymerase alpha (Pol α) and primase – to initiate DNA synthesis by polymerisation of an RNA–DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo‐electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation. Eukaryotic DNA replication depends on the primosome, a protein complex that initiates DNA synthesis by polymerisation of an RNA–DNA primer. Our research has captured the conformational changes of the human primosome under ideal biochemical conditions. Using cryoEM, we have successfully revealed the structural intermediates of the active primosome, showcasing the synchronised movements of its components PRIM1, PRIM2, and POLA1 in RNA primer synthesis. This breakthrough contributes to a comprehensive understanding of the dynamic structure of the human primosome, enhancing our grasp of the intricacies of DNA replication.