Molecular basis for the initiation of DNA primer synthesis

During the initiation of DNA replication, oligonucleotide primers are synthesised de novo by primases, which are subsequently extended by replicative polymerases to complete genome duplication. The Primase-Polymerase (Prim- Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-Associated Primase-Polymerases (CAPPs) involved in adaptive immunity1–3. While much is known about the activities of these enzymes, the precise mechanism utilised by primases to initiate primer synthesis has not been elucidated. Here we reveal the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors, and paired to the templating ssDNA strand, prior to the synthesis of the first phosphodiester bond. Furthermore, a Prim-Pol complex with dsDNA reveals how the enzyme subsequently extends the primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol’s instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. It also establishes that the catalytic domain of Prim-Pols, including replicative primases, is sufficient to catalyse primer formation.